Biological control of cucumber green mottle mosaic virus

ABSTRACT

An attenuated strain of cucumber green mottle mosaic virus (CGMMV) is useful to protect cucumber plants from infection with the wild-type infectious CGMMV strain. The genome of the attenuated virus contains at least one mutation or group of mutations selected from c.4969G&gt;A, c.3334C&gt;T, and a group of at least six of the mutations c.315G&gt;A; c.1498A&gt;G; c.1660C&gt;T; c.3430C&gt;T; c.3528A&gt;G; c.4144C&gt;T; c.4248C&gt;T; and c.6228C&gt;T. These mutated genomes encode one or more mutations selected from R1637H in the 186 kDa readthrough replication protein, A1092V in the 129 kDa replication protein and/or the 186 kDa readthrough replication protein, and at least six mutations selected from G86S, E480G, S534F, A1124V, N1157D, P1362L, P1397S in the 129 kDa replication protein and/or the 186 kDa readthrough replication protein, and the A156V mutation in the coat protein.

CROSS REFERENCE TO A RELATED APPLICATION

This application claims priority to Canadian Patent Application No.3,017,465, filed Sep. 14, 2018, which is incorporated herein byreference in its entirety.

The Sequence Listing for this application is labeled“SeqList-31Aug19-ST25.txt”, which was created on Sep. 14, 2018, and is290 KB. The entire content is incorporated herein by reference in itsentirety.

FIELD

The present application is directed to attenuated plant viruses. Morespecifically, the present application provides attenuated strains ofcucumber green mottle mosaic virus (CGMMV), compositions thereof, andmethods of using such virus strains and compositions for biologicalcontrol of plant disease.

BACKGROUND

Cucumber green mottle mosaic virus (CGMMV) is a member of theTobamovirus genus in the family Virgaviridae. CGMMV has a 6.4-kbsingle-stranded, positive-sense RNA genome with a 3′ tRNA-like structureinstead of a poly(A) tail. The genome contains three open reading frames(ORFs) that encode four defined proteins. The first ORF encodes a 129kDa protein, including methyltransferase and helicase domains requiredfor RNA replication, and a 186 kDa protein produced by readthroughtranslation of the ORF, including the methyltransferase and helicasedomains and an additional RNA-dependent RNA polymerase (RdRp) domain.The remaining two ORFs encode a movement protein (MP) and a coat protein(CP), respectively.

CGMMV infection causes serious diseases in plants of the familyCucurbitaceae (cucurbits), including cucumber, pumpkin, watermelon,melon, squash, zucchini, gourds, gherkins and others. CGMMV was firstreported in 1935 in the UK and is found in Europe, Asia, the Middle Eastand, since 2013, in Canada and the United States. CGMMV infection isbecoming a major limiting factor with regard to production of cucurbitsworldwide, and has become an increasing threat to the commercialproduction of cucumber and other commercially-grown cucurbit crops.

CGMMV is a seed-borne virus and can be transmitted by root-to-rootcontact and by transfer from contaminated seeds, soil, gardeningimplements such as pruners and stakes, irrigation water, packingmaterials, or the clothing or hands of farmers, pickers or other personshandling the plants. The CGMMV virus is extremely stable and can remaininfectious under relatively extreme conditions for long periods of time.Therefore, the presence of even a few infected plants in a cucumbergreenhouse can eventually lead to the spread of CGMMV infection to theentire crop.

CGMMV is responsible for a wide range of symptoms on leaves and fruitsof infected plants, depending on the CGMMV strain, stage of infectionand plant species. The induced symptoms include vein clearing andcrumpling in young leaves, light green mottling, mosaic patterns,necrotic lesions, fruit distortion or streak, change of sugaraccumulation and flavor, and premature degradation of the pulp, makingthe fruit unmarketable and unfit for consumption. CGMMV infection canresult in substantial yield losses up to 100%, although losses of 40-80%are more common in a commercial field or greenhouse production setting,in addition to having a major impact on fruit quality, leading to lowmarket value.

There are no known effective chemical methods for controlling virusdiseases of plants, and crop protection relies completely on aspects ofsanitation that may include removal of infected plants, using onlyvirus-free seeds and vegetative stocks, using resistant varieties, andcontrolling transmission by contact with insects, water and humans. Twonew techniques that are now being investigated for managing plant virusdiseases rely on cross protection by inoculation with attenuated/mildstrains and by creating transgenic or engineered resistance based onexpression of the viral coat protein of a specific viral pathogen.

Cross protection is an acquired immunity phenomenon which has beendemonstrated in a number of systems. The technique makes use of theobservation that plants infected with an attenuated virus isolate orstrain causing mild or no symptoms can develop tolerance to furtherinfection when challenged by an isolate or strain of the same or arelated virus species which causes more severe symptoms. Crossprotection is virus specific, occurring only between strains of the samevirus or related virus species, and is seen as an acceptable method forcrop protection in greenhouse systems as it does not rely on harmfulmaterials or chemicals. This approach was first applied successfully inseveral countries in the 1970s to protect tomato plants againstinfection with tobacco mosaic virus. More recently, in 2015, thetreatment PMV™-01, which contains a mild isolate of the Chilean strainof pepino mosaic virus (PepMV), has been registered in Europe andcommercially used to protect tomato plants from severe losses in qualityand yield caused by aggressive infection with PepMV.

In order to develop cross protection, attenuated/mild strains of a plantvirus are needed which cause no visible or only very mild symptoms butwhich prevent infection by strains causing more severe symptoms.Attenuated isolates advantageously have little or no impact on plantsymptoms, total yield and fruit quality, and effectively protect againstmore virulent isolates. The following properties have been proposed asdesirable criteria for an attenuated virus strain for use in cropprotection:

-   -   no symptoms or very mild symptoms are induced in any of the        cultivated hosts, and the quality and quantity of the crop        products are not reduced;    -   most host tissues are fully infected systemically;    -   the attenuated strains are highly stable genetically without        mutating into a severe phenotype;    -   there is no vector transmission to other crops;    -   the attenuated strains protect against a wide range of viruses        and strains; and    -   the quality/quantity control of the inoculum and inoculations        are easy and inexpensive.

An attenuated CGMMV strain SH33b is known (Motoyoshi, F., andNishiguchi, M. 1988. Control of virus diseases by attenuated virusstrains: comparison between attenuated strains of cucumber green mottlemosaic virus and tobacco mosaic virus. Gamma Field Symposia, 27: 91-107)which induces no or only mild systematic symptoms in leaves of muskmelonplants when a low concentration of attenuated virus was used toinoculate muskmelon seedlings. However, although this strain waseffective in protecting muskmelon plants from outbreaks of severesymptoms, and in eliminating wild-type CGMMV from the greenhouse,inoculation of cotyledons with higher concentrations of the purifiedvirus led to appearance of mosaic symptoms in the upper leaves.

In addition, an attenuated CGMMV strain VIROG-43M is known(Slavokhotova, A. A., et al, American Journal of Plant Sciences (2016),7: 724-732), which showed effectiveness in protecting inoculatedcucumber plants from disease symptoms under greenhouse conditions.However, VIROG-43M itself could induce symptoms in inoculated cucumberplants after two months of inoculation. In addition, only 85% of thecucumber plants inoculated with VIROG-43M and challenged with thepathogenic CGMMV strain NC-1 were observed to be symptomless after 3months of inoculation under laboratory greenhouse conditions.

Recently, it was reported that several attenuated CGMMV strains wereobtained from a pathogenic CGMMV strain by multi site-directedmutagenesis, based on sequence comparison of related tobamoviruses(Chen, Bin, “Molecular Characterization of Viruses Infecting GreenhouseVegetables in Ontario” (2016). Electronic Thesis and DissertationRepository. 4222, University of Western Ontario. Available athttps://ir.lib.uwo.ca/etd/4222). However, the reported mutants coulddevelop visible mosaic symptoms in cucumber plants to various extents,although the symptoms were generally mild compared with those caused bythe wild-type strain. Even the best attenuated CGMMV isolates inducedsymptoms in cucumber plants after 28 days post inoculation.

It is therefore desirable to provide an attenuated strain of CGMMV forprotecting plants against infection with CGMMV, and in particular, forprotecting plants which are susceptible to infection with CGMMV such asspecies of the Cucurbitaceae family.

SUMMARY

In one aspect, the present application provides an attenuated strain ofcucumber green mottle mosaic virus (CGMMV) comprising a genome, whereinthe genome is a polyribonucleotide having a sequence functionallyequivalent to a variant of SEQ ID NO: 18, the sequence comprising atleast one residue or group of residues selected from:

a) A at the position corresponding to position 4969 of SEQ ID NO:18;

b) U at the position corresponding to position 3334 of SEQ ID NO: 18;and

c) a group of at least six residues selected from:

A at the position corresponding to position 315 of SEQ ID NO: 18;

G at the position corresponding to position 1498 of SEQ ID NO: 18;

U at the position corresponding to position 1660 of SEQ ID NO: 18;

U at the position corresponding to position 3430 of SEQ ID NO:18;

G at the position corresponding to position 3528 of SEQ ID NO: 18;

U at the position corresponding to position 4144 of SEQ ID NO:18;

U at the position corresponding to position 4248 of SEQ ID NO: 18; and

U at the position corresponding to position 6228 of SEQ ID NO: 18.

In another aspect, the present application provides apolydeoxyribonucleotide having a sequence functionally equivalent to asequence of a cucumber green mottle mosaic virus (CGMMV) genome, whereinthe sequence of the polydeoxyribonucleotide is a variant of SEQ ID NO:18 comprising at least one residue or group of residues selected from:

a) A at the position corresponding to position 4969 of SEQ ID NO: 18;

b) T at the position corresponding to position 3334 of SEQ ID NO: 18;and

c) a group of at least six residues selected from:

A at the position corresponding to position 315 of SEQ ID NO:18;

G at the position corresponding to position 1498 of SEQ ID NO:18;

T at the position corresponding to position 1660 of SEQ ID NO: 18;

T at the position corresponding to position 3430 of SEQ ID NO: 18;

G at the position corresponding to position 3528 of SEQ ID NO: 18;

T at the position corresponding to position 4144 of SEQ ID NO:18;

T at the position corresponding to position 4248 of SEQ ID NO:18; and

T at the position corresponding to position 6228 of SEQ ID NO: 18.

In at least one embodiment, the polydeoxyribonucleotide is configuredfor expression in a host cell. In at least one embodiment, the host cellis a microorganism. In at least one embodiment, the host cell is a plantcell.

In another aspect, the present application provides a vector comprisinga polydeoxyribonucleotide as described herein. In at least oneembodiment, the vector is configured to genetically modify a host cell.In at least one embodiment, the host cell is a microorganism. In atleast one embodiment, the host cell is a plant cell.

A further aspect of the present application provides a geneticallymodified cell comprising a polydeoxyribonucleotide as described herein.In at least one embodiment, the cell is a microorganism. In at least oneembodiment, the cell is a plant cell.

Yet another aspect of the present application provides a composition forpreventing symptoms associated with infection by wild-type CGMMV in aplant, where the composition comprises an attenuated strain of CGMMV ora genetically modified cell as described herein and an agriculturallyacceptable carrier.

In an additional aspect, the present application provides a compositionfor increasing resistance of a plant to infection by wild-type CGMMV,where the composition comprises an attenuated strain of CGMMV or agenetically modified cell as described herein and an agriculturallyacceptable carrier.

Another aspect of the present application provides a method forpreventing symptoms associated with infection by wild-type CGMMV in aplant, where the method includes inoculating the plant with anattenuated strain of CGMMV or with a genetically modified cell or with acomposition as described herein.

A further aspect of the present application provides a method forincreasing resistance of a plant to infection by wild-type CGMMV, wherethe method includes inoculating the plant with an attenuated strain ofCGMMV or with a genetically modified cell or with a composition asdescribed herein.

In a further aspect, the present application provides a geneticallymodified plant comprising a genome which comprises apolydeoxyribonucleotide as described herein. In at least one embodiment,the plant is a cucurbit. In at least one embodiment, the plant is acucumber plant.

BRIEF DESCRIPTION OF THE DRAWINGS

Further features of the present invention will become apparent from thefollowing written description and the accompanying figures, in which:

FIG. 1A is a photograph showing leaves of a cucumber plant grown for twoweeks after inoculation with a clone of a wild-type cucumber greenmottle mosaic virus (CGMMV) Ontario strain;

FIG. 1B is a photograph showing leaves of a cucumber plant grown for twoweeks after inoculation with a wild-type CGMMV Ontario strain isolate;

FIG. 1C is a photograph showing leaves of a cucumber plant grown for twoweeks in the absence of CGMMV (negative control);

FIG. 2A is a photograph showing leaves of a cucumber plant infected withwild-type CGMMV Ontario strain and grown under laboratory conditions;

FIG. 2B is a photograph showing leaves of a cucumber plant infected withthe mutant CGMMV Ontario strain ONB and grown under the laboratoryconditions of FIG. 2A;

FIG. 2C is photograph showing leaves of a cucumber plant infected withthe mutant CGMMV Ontario strain ONM and grown under the laboratoryconditions of FIG. 2A;

FIG. 2D is a photograph showing leaves of a healthy cucumber plantwithout CGMMV infection grown as a control under the laboratoryconditions of FIG. 2A;

FIG. 3A is a photograph showing leaves of a cucumber plant infected withwild-type CGMMV Ontario strain and grown under laboratory conditions;

FIG. 3B is a photograph showing leaves of a healthy cucumber plantwithout CGMMV infection grown as a control under the laboratoryconditions of FIG. 3A;

FIG. 3C is a photograph showing leaves of a cucumber plant exposed to anattenuated CGMMV strain according to the present invention (ONBM) andgrown under the laboratory conditions of FIG. 3A;

FIG. 3D is a photograph showing leaves of a cucumber plant exposed toanother attenuated CGMMV strain according to the present invention(ONBM-2) and grown under the laboratory conditions of FIG. 3A;

FIG. 3E is a photograph showing leaves of a cucumber plant exposed toyet another attenuated CGMMV strain according to the present invention(ONBM-3) and grown under the laboratory conditions of FIG. 3A;

FIG. 4A is a photograph showing leaves of a cucumber plant inoculatedwith an attenuated CGMMV strain according to the present invention(ONBM), then challenged with a wild-type CGMMV and grown underlaboratory conditions;

FIG. 4B is a photograph showing leaves of a cucumber plant inoculatedwith another attenuated CGMMV strain according to the present invention(ONBM-2), then challenged with a wild-type CGMMV and grown under thelaboratory conditions of FIG. 4A;

FIG. 4C is a photograph showing leaves of a cucumber plant inoculatedwith yet another attenuated CGMMV strain according to the presentinvention (ONBM-3), then challenged with a wild-type CGMMV and grownunder the laboratory conditions of FIG. 4A;

FIG. 4D is a photograph showing leaves of a cucumber plant infected withwild-type CGMMV and grown under the laboratory conditions of FIG. 4A;

FIG. 4E is a photograph showing leaves of a healthy cucumber plantwithout CGMMV infection grown as a control under the laboratoryconditions of FIG. 4A;

FIG. 5A is a photograph showing leaves of a cucumber plant grown in acommercial greenhouse and infected with a wild-type CGMMV Ontariostrain;

FIG. 5B is a photograph showing a mosaic pattern on fruit of a cucumberplant grown in the commercial greenhouse of FIG. 5A and infected with awild-type CGMMV Ontario strain;

FIG. 5C is a photograph showing curling of fruit of a cucumber plantgrown in the commercial greenhouse of FIG. 5A and infected with awild-type CGMMV Ontario strain;

FIG. 5D is a photograph showing leaves and fruit of a cucumber plantexposed to an attenuated CGMMV strain according to the present invention(ONBM-2) and grown in the commercial greenhouse of FIG. 5A;

FIG. 5E is a photograph showing leaves and fruit of a cucumber plantexposed to another attenuated CGMMV strain according to the presentinvention (ONBM-3) and grown in the commercial greenhouse of FIG. 5A;

FIG. 6A is a photograph showing leaves of a cucumber plant exposed toanother attenuated CGMMV strain according to the present invention(ONAL-1) and grown under laboratory conditions;

FIG. 6B is a photograph showing leaves of a cucumber plant exposed toanother attenuated CGMMV strain according to the present invention(ONAL-2) and grown under the laboratory conditions of FIG. 6A;

FIG. 6C is a photograph showing leaves of a cucumber plant exposed toanother attenuated CGMMV strain according to the present invention(ONBM-32) and grown under the laboratory conditions of FIG. 6A;

FIG. 6D is a photograph showing leaves of a cucumber plant exposed tothe mutant CGMMV Ontario strain ONB and grown under the laboratoryconditions of FIG. 6A;

FIG. 6E is a photograph showing leaves of a healthy cucumber plantwithout CGMMV infection grown as a control under the laboratoryconditions of FIG. 6A; and

FIG. 6F is a photograph showing leaves of a cucumber plant infected withwild-type CGMMV Ontario strain and grown under the laboratory conditionsof FIG. 6A.

DETAILED DESCRIPTION

The present application provides an attenuated strain of the cucumbergreen mottle mosaic virus (CGMMV). In at least one embodiment, theattenuated CGMMV strain may be useful for protecting one or more plantsagainst the deleterious effects of infection by a wild-type strain ofCGMMV. In at least one embodiment, plants which may be protected againstinfection by wild-type CGMMV include but are not limited to any plantsusceptible to infection by CGMMV, including but not limited to plantsof the family Cucurbitaceae (cucurbits), including but not limited tovarieties of cucumber (Cucumis sativus), pumpkin, watermelon, melon,squash, zucchini, gourds, gherkins and others well known in the art. Inat least one embodiment, curcubits such as cucumber plants inoculatedwith at least one embodiment of the attenuated CGMMV strain may not showsignificant visible symptoms for a period of up to two months or longerafter inoculation with the attenuated CGMMV strain.

In at least one embodiment, the attenuated strain of CGMMV has a genomewhich is a polyribonucleotide having a sequence which is functionallyequivalent to a variant of SEQ ID NO: 18 including one or morevariations thereof.

In at least one embodiment, the polyribonucleotide has a sequenceincluding A at the position corresponding to position 4969 of SEQ ID NO:18.

In at least one embodiment, the polyribonucleotide has a sequenceincluding U at the position corresponding to position 3334 of SEQ ID NO:18.

In at least one embodiment, the polyribonucleotide has a sequenceincluding A at the position corresponding to position 4969 of SEQ ID NO:18 and U at the position corresponding to position 3334 of SEQ ID NO:18.

In at least one embodiment, the polyribonucleotide has a sequenceincluding at least six of the following residues:

A at the position corresponding to position 315 of SEQ ID NO: 18;

G at the position corresponding to position 1498 of SEQ ID NO:18;

U at the position corresponding to position 1660 of SEQ ID NO: 18;

U at the position corresponding to position 3430 of SEQ ID NO: 18;

G at the position corresponding to position 3528 of SEQ ID NO: 18;

U at the position corresponding to position 4144 of SEQ ID NO:18;

U at the position corresponding to position 4248 of SEQ ID NO: 18; and

U at the position corresponding to position 6228 of SEQ ID NO: 18.

In at least one such embodiment, the polyribonucleotide sequence alsoincludes one or both of:

U at the position corresponding to position 3334 of SEQ ID NO: 18; and

A at the position corresponding to position 4969 of SEQ ID NO: 18.

In at least one embodiment, the polyribonucleotide has a sequenceincluding the following residues:

A at the position corresponding to position 315 of SEQ ID NO: 18;

G at the position corresponding to position 1498 of SEQ ID NO:18;

U at the position corresponding to position 1660 of SEQ ID NO: 18;

U at the position corresponding to position 3430 of SEQ ID NO: 18;

G at the position corresponding to position 3528 of SEQ ID NO: 18;

U at the position corresponding to position 4144 of SEQ ID NO:18;

U at the position corresponding to position 4248 of SEQ ID NO: 18; and

U at the position corresponding to position 6228 of SEQ ID NO: 18.

In at least one such embodiment, the polyribonucleotide sequence alsoincludes one or both of:

U at the position corresponding to position 3334 of SEQ ID NO: 18; and

A at the position corresponding to position 4969 of SEQ ID NO: 18.

As used herein, the term “polynucleotide” is intended to mean apolymeric molecule comprising two or more nucleosides linked throughcovalent bonds to phosphate groups, such that the 5′-hydroxyl group of anucleoside and the 3′-hydroxyl group of an adjacent nucleoside are bothcovalently bonded to the same phosphate group. As understood in the art,when covalently linked together to form the polynucleotide molecule,each individual nucleoside unit is also known as a “residue”. As usedherein, the term “nucleoside” is intended to mean a molecule in which asugar moiety selected from ribose and deoxyribose is bonded to a purineor pyrimidine base moiety selected from adenine (A), guanine (G),cytosine (C), thymine (T) or uracil (U). A polynucleotide includespolynucleotides of any length, including but not limited todinucleotides, trinucleotides, tetranucleotides, oligonucleotides andnucleic acids.

When the sugar moiety is ribose, the base is selected from A, G, C andU, and the nucleoside is referred to as a ribonucleoside. Apolynucleotide comprising two or more such ribonucleosides is referredto as a polyribonucleotide or ribonucleic acid (RNA). When the sugarmoiety is deoxyribose, the base is selected from A, G, C and T, and thenucleoside is referred to as a deoxyribonucleoside. A polynucleotidecomprising two or more such deoxyribonucleosides is referred to as apolydeoxyribonucleotide or deoxyribonucleic acid (DNA).

In at least one embodiment, the attenuated strains can be obtained bymutation of wild type CGMMV to introduce one or more variations ormutations into the genome sequence of the wild type CGMMV. As usedherein interchangeably with respect to a polynucleotide sequence, theterm “variation” or “mutation” is intended to refer to a difference inthe polynucleotide sequence with respect to a reference polynucleotidesequence. Variations or mutations can include substitution of one ormore nucleotide residues with different nucleotide residues, insertionof additional nucleotide residues or deletion of nucleotide residues. Avariation or mutation may or may not alter the open reading frame(s) ofthe polynucleotide or the amino acid sequence of any protein(s) encodedby the polynucleotide. In at least one embodiment, the variation ormutation is a naturally occurring variation or mutation arising withoutartificial intervention. In at least one embodiment, the variation ormutation is introduced intentionally by methods well known in the art,including but not limited to random mutagenesis or directed mutagenesis.

An RNA virus such as CGMMV, including but not limited to attenuatedstrains thereof, contains an RNA genome, in which the geneticinformation of the virus is stored in the form of RNA. As will beunderstood by a person of skill in the art, it is possible to expressviral proteins encoded by such an RNA genome in a host cell whichexpresses genetic information from DNA by preparing a complementary DNA(cDNA) molecule carrying the same genetic information as is encoded bythe RNA genome, and transforming the host cell with the cDNA such thatthe transformed host cell can express viral proteins encoded by thecDNA. In at least one embodiment, the virus can be assembled and/orreplicated within the transformed host cell.

Therefore, another aspect of the present invention provides apolydeoxyribonucleotide having a sequence functionally equivalent to asequence of a cucumber green mottle mosaic virus (CGMMV) genome, whereinthe polydeoxyribonucleotide sequence comprises a variant of SEQ ID NO:18 including one or more variations from SEQ ID NO: 18.

In at least one embodiment, the polydeoxyribonucleotide sequencecomprises a sequence wherein the one or more variations from SEQ ID NO:18 include A at the position corresponding to position 4969 of SEQ IDNO: 18.

In at least one embodiment, the polydeoxyribonucleotide sequencecomprises a sequence wherein the one or more variations from SEQ ID NO:18 include T at the position corresponding to position 3334 of SEQ IDNO: 18.

In at least one embodiment, the polydeoxyribonucleotide sequencecomprises a sequence wherein the one or more variations from SEQ ID NO:18 include A at the position corresponding to position 4969 of SEQ IDNO: 18 and T at the position corresponding to position 3334 of SEQ IDNO:18.

In at least one embodiment, the polydeoxyribonucleotide sequencecomprises a sequence wherein the one or more variations from SEQ IDNO:18 include at least six of the following residues:

A at the position corresponding to position 315 of SEQ ID NO: 18;

G at the position corresponding to position 1498 of SEQ ID NO:18;

T at the position corresponding to position 1660 of SEQ ID NO:18;

T at the position corresponding to position 3430 of SEQ ID NO:18;

G at the position corresponding to position 3528 of SEQ ID NO: 18;

T at the position corresponding to position 4144 of SEQ ID NO: 18;

T at the position corresponding to position 4248 of SEQ ID NO: 18; and

T at the position corresponding to position 6228 of SEQ ID NO: 18.

In at least one such embodiment, the one or more variations from SEQ IDNO: 18 further include one or both of:

T at the position corresponding to position 3334 of SEQ ID NO:18; and

A at the position corresponding to position 4969 of SEQ ID NO: 18.

In at least one embodiment, the polydeoxyribonucleotide sequencecomprises a sequence wherein the one or more variations from SEQ ID NO:18 include the following residues:

A at the position corresponding to position 315 of SEQ ID NO: 18;

G at the position corresponding to position 1498 of SEQ ID NO: 18;

T at the position corresponding to position 1660 of SEQ ID NO:18;

T at the position corresponding to position 3430 of SEQ ID NO: 18;

G at the position corresponding to position 3528 of SEQ ID NO: 18;

T at the position corresponding to position 4144 of SEQ ID NO: 18;

T at the position corresponding to position 4248 of SEQ ID NO: 18; and

T at the position corresponding to position 6228 of SEQ ID NO: 18.

In at least one such embodiment, the one or more variations from SEQ IDNO: 18 further include one or both of:

T at the position corresponding to position 3334 of SEQ ID NO: 18; and

A at the position corresponding to position 4969 of SEQ ID NO:18.

As used herein with reference to a polynucleotide sequence, the term“functionally equivalent to” is intended to mean that the polynucleotidesequence contains the same genetic information, including but notlimited to coding information, as the genetic information contained inthe reference polynucleotide sequence. In at least one embodiment, afunctionally equivalent polynucleotide sequence will encode the sameprotein or proteins as are encoded by the reference polynucleotidesequence. In at least one embodiment, a given position in thepolynucleotide sequence will bear a base equivalent to the base borne bythe corresponding position of the reference polynucleotide sequence. Asused herein with reference to bases in functionally equivalentpolynucleotide sequences, the term “equivalent” is intended to mean thatthe bases are either identical or provide the same coding information.Thus, the base T (which is found in polydeoxyribonucleotides) and thebase U (which is found in polyribonucleotides) are considered herein tobe equivalent to each other. In other words, when apolydeoxyribonucleotide sequence is functionally equivalent to apolyribonucleotide sequence, positions in the polydeoxyribonucleotidesequence which bear the base T are considered to correspond to positionsin the polyribonucleotide sequence which bear the base U.

In at least one embodiment, the sequence of the polydeoxyribonucleotideis selected from SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:53,SEQ ID NO:58, SEQ ID NO:60, and variants thereof which include at leastone residue or group of residues selected from:

a) A at the position corresponding to position 4969 of SEQ ID NO: 18;

b) T at the position corresponding to position 3334 of SEQ ID NO: 18;and

c) a group of at least six residues selected from:

A at the position corresponding to position 315 of SEQ ID NO: 18;

G at the position corresponding to position 1498 of SEQ ID NO:18;

T at the position corresponding to position 1660 of SEQ ID NO:18;

T at the position corresponding to position 3430 of SEQ ID NO: 18;

G at the position corresponding to position 3528 of SEQ ID NO: 18;

T at the position corresponding to position 4144 of SEQ ID NO:18;

T at the position corresponding to position 4248 of SEQ ID NO:18; and

T at the position corresponding to position 6228 of SEQ ID NO: 18.

In at least one embodiment, the sequence of the variant can have atleast 89%, at least 90%, at least 95%, at least 99% or at least 99.9%identity to a sequence selected from SEQ ID NO:42, SEQ ID NO:45, SEQ IDNO:48, SEQ ID NO:53, SEQ ID NO:58 or SEQ ID NO:60, wherein the variantsequence comprises at least one residue or group of residues selectedfrom:

a) A at the position corresponding to position 4969 of SEQ ID NO:18;

b) T at the position corresponding to position 3334 of SEQ ID NO: 18;and

c) a group of at least six residues selected from:

A at the position corresponding to position 315 of SEQ ID NO: 18;

G at the position corresponding to position 1498 of SEQ ID NO:18;

T at the position corresponding to position 1660 of SEQ ID NO: 18;

T at the position corresponding to position 3430 of SEQ ID NO: 18;

G at the position corresponding to position 3528 of SEQ ID NO: 18;

T at the position corresponding to position 4144 of SEQ ID NO:18;

T at the position corresponding to position 4248 of SEQ ID NO: 18; and

T at the position corresponding to position 6228 of SEQ ID NO: 18.

In at least one embodiment, the sequence of the polydeoxyribonucleotideis selected from SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:53,SEQ ID NO:58, SEQ ID NO:60, and variants thereof which encode one ormore of the proteins encoded by the polydeoxyribonucleotide having thesequence selected from SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ IDNO:53, SEQ ID NO:58 and SEQ ID NO:60.

As used herein, the term “variant” when used in reference to apolynucleotide is intended to refer to a polynucleotide which differs inits nucleotide sequence from the sequence of a reference polynucleotideto which the variant is being compared by one or more nucleotideresidues. The differences between the sequence of the variant and thesequence of the reference polynucleotide, also referred to herein asvariations or mutations, can include substitution of one or morenucleotide residues with different nucleotide residues, insertion ofadditional nucleotide residues or deletion of nucleotide residues. Incertain embodiments, a variant can differ from a referencepolynucleotide by substitution of one or more nucleotide residues withreplacement nucleotide residues which do not alter the open readingframe(s) of the polynucleotide or the amino acid sequence of anyprotein(s) encoded by the polynucleotide.

As used herein, the term “variant” when used in reference to apolypeptide is intended to refer to a polypeptide which differs in itsamino acid sequence from the sequence of a reference polypeptide towhich the variant is being compared by one or more amino acid residues.The differences between the sequence of the variant and the sequence ofthe reference polypeptide can include substitution of one or more aminoacid residues with different amino acid residues, insertion ofadditional amino acid residues or deletion of amino acid residues. Incertain embodiments, a variant can differ from a reference polypeptideby conservative substitution of one or more amino acid residues withreplacement amino acid residues which may have similar properties,including but not limited to charge, size and hydrophilicity, to theamino acid residues which the new residues replace. In certainembodiments, variants may completely or partially retain one or morebiological functions of the reference polypeptide. In certainembodiments, variants may not retain one or more biological functions ofthe reference polypeptide.

As used herein, the term “percent identity” or “% identity” when used inreference to the sequence of a polypeptide or a polynucleotide isintended to mean the percentage of the total number of amino acid ornucleotide residues, respectively, in the sequence which are identicalto those at the corresponding position of a reference polypeptide orpolynucleotide sequence. In at least one embodiment, when the length ofthe variant sequence and the length of the reference sequence are notidentical, percent identity can be calculated based on the total numberof residues in the variant sequence or based on the total number orresidues in the reference sequence. Percent identity can be measured byvarious local or global sequence alignment algorithms well known in theart, including but not limited to the Smith-Waterman algorithm and theNeedleman-Wunsch algorithm. Tools using these or other suitablealgorithms include but are not limited to BLAST (Basic Local AlignmentSearch Tool) and other such tools well known in the art.

In at least one embodiment, a variant polynucleotide sequence canhybridize to a polyribonucleotide or polydeoxyribonucleotide asdescribed herein under at least moderately stringent conditions. By “atleast moderately stringent hybridization conditions” it is meant thatconditions are selected which promote selective hybridization betweentwo complementary nucleic acid molecules in solution. Hybridization mayoccur to all or a portion of a nucleic acid sequence molecule. Thehybridizing portion is typically at least 15 (e.g. 20, 25, 30, 40 or 50)nucleotides in length. Those skilled in the art will recognize that thestability of a nucleic acid duplex, or hybrid, is determined by themelting temperature (T_(m)), which in sodium-containing buffers is afunction of the sodium ion concentration ([Na⁺]) and temperature(T_(m)=81.5° C. −16.6 (Log₁₀ [Na⁺])+0.41(% (G+C)−600/I), where % G+C isthe percentage of cytosine and guanine nucleotides in the nucleic acidand 1 is the length of the nucleic acid in base pairs, or similarequation). Accordingly, the parameters in the wash conditions thatdetermine hybrid stability are sodium ion concentration and temperature.In order to identify molecules that are similar, but not identical, to aknown nucleic acid molecule, a 1% mismatch may be assumed to result inabout a 1° C. decrease in T_(m). For example, if nucleic acid moleculesare sought that have a >95% identity, the final wash temperature may bereduced by about 5° C. Based on these considerations those skilled inthe art will be able to readily select appropriate hybridizationconditions.

In some embodiments, stringent hybridization conditions are selected. Byway of example the following conditions may be employed to achievestringent hybridization: hybridization at 5× sodium chloride/sodiumcitrate (SSC)/5×Denhardt's solution/1.0% sodium dodecylsulfate (SDS) atT_(m)−5° C. based on the above equation, followed by a wash of0.2×SSC/0.1% SDS at 60° C. Moderately stringent hybridization conditionsinclude a washing step in 3×SSC at 42° C. It is understood, however,that equivalent stringencies may be achieved using alternative buffers,salts and temperatures. Additional guidance regarding hybridizationconditions may be found in: Current Protocols in Molecular Biology, JohnWiley & Sons, N.Y., 2002, and in: Sambrook et al., Molecular Cloning: aLaboratory Manual, Cold Spring Harbor Laboratory Press, 2001.

In at least one embodiment, the polyribonucleotide CGMMV genome and thefunctionally equivalent polydeoxyribonucleotide can each encode one ormore viral proteins including but not limited to a 129 kDa proteinincluding methyltransferase and helicase domains required for RNAreplication, a 186 kDa protein including the methyltransferase andhelicase domains and an additional RNA-dependent RNA polymerase (RdRp)domain, a movement protein (MP) and a coat protein (CP).

In at least one embodiment, the 129 kDa protein has an amino acidsequence comprising valine (V, Val) at the position corresponding toposition 1092 of SEQ ID NO:63.

In at least one embodiment, the 129 kDa protein sequence includes atleast two residues selected from:

-   -   serine (S, Ser) at the position corresponding to position 86 of        SEQ ID NO:63;    -   glycine (G, Gly) at the position corresponding to position 480        of SEQ ID NO:63;    -   phenylalanine (F, Phe) at the position corresponding to position        534 of SEQ ID NO:63; and    -   valine (V, Val) at the position corresponding to position 1124        of SEQ ID NO:63.

In at least one such embodiment, the 129 kDa protein sequence furtherincludes valine (V, Val) at the position corresponding to position 1092of SEQ ID NO:63.

In at least one embodiment, the 129 kDa protein sequence includes atleast three residues selected from:

-   -   serine (S, Ser) at the position corresponding to position 86 of        SEQ ID NO:63;    -   glycine (G, Gly) at the position corresponding to position 480        of SEQ ID NO:63;    -   phenylalanine (F, Phe) at the position corresponding to position        534 of SEQ ID NO:63; and    -   valine (V, Val) at the position corresponding to position 1124        of SEQ ID NO:63.

In at least one such embodiment, the 129 kDa protein sequence furtherincludes valine (V, Val) at the position corresponding to position 1092of SEQ ID NO:63.

In at least one embodiment, the 129 kDa protein sequence includes theresidues:

-   -   serine (S, Ser) at the position corresponding to position 86 of        SEQ ID NO:63;    -   glycine (G, Gly) at the position corresponding to position 480        of SEQ ID NO:63;    -   phenylalanine (F, Phe) at the position corresponding to position        534 of SEQ ID NO:63; and    -   valine (V, Val) at the position corresponding to position 1124        of SEQ ID NO:63.

In at least one such embodiment, the 129 kDa protein sequence furtherincludes valine (V, Val) at the position corresponding to position 1092of SEQ ID NO:63.

In at least one embodiment, the sequence of the 129 kDa protein isselected from SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:54,SEQ ID NO:61 and variants thereof which include valine (V, Val) at theposition corresponding to position 1092 of SEQ ID NO:63.

In at least one embodiment, the sequence of the 129 kDa protein isselected from SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:54,SEQ ID NO:61 and variants thereof which include at least two residuesselected from:

-   -   serine (S, Ser) at the position corresponding to position 86 of        SEQ ID NO:63;    -   glycine (G, Gly) at the position corresponding to position 480        of SEQ ID NO:63;    -   phenylalanine (F, Phe) at the position corresponding to position        534 of SEQ ID NO:63; and    -   valine (V, Val) at the position corresponding to position 1124        of SEQ ID NO:63.

In at least one such embodiment, the variants of the 129 kDa proteinsequence further include valine (V, Val) at the position correspondingto position 1092 of SEQ ID NO:63.

In at least one embodiment, the sequence of the 129 kDa protein isselected from SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:54,SEQ 1D NO:61 and variants thereof which include at least three residuesselected from:

-   -   serine (S, Ser) at the position corresponding to position 86 of        SEQ ID NO:63;    -   glycine (G, Gly) at the position corresponding to position 480        of SEQ ID NO:63;    -   phenylalanine (F, Phe) at the position corresponding to position        534 of SEQ ID NO:63; and    -   valine (V, Val) at the position corresponding to position 1124        of SEQ ID NO:63.

In at least one such embodiment, the variants of the 129 kDa proteinsequence further include valine (V, Val) at the position correspondingto position 1092 of SEQ ID NO:63.

In at least one embodiment, the sequence of the 129 kDa protein isselected from SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:54,SEQ ID NO:61 and variants thereof which include the residues:

-   -   serine (S, Ser) at the position corresponding to position 86 of        SEQ ID NO:63;    -   glycine (G, Gly) at the position corresponding to position 480        of SEQ ID NO:63;    -   phenylalanine (F, Phe) at the position corresponding to position        534 of SEQ ID NO:63; and    -   valine (V, Val) at the position corresponding to position 1124        of SEQ ID NO:63.

In at least one such embodiment, the variants of the 129 kDa proteinsequence further include valine (V, Val) at the position correspondingto position 1092 of SEQ ID NO:63.

In at least one embodiment, the 186 kDa protein has an amino acidsequence comprising histidine (H, His) at the position corresponding toposition 1637 of SEQ ID NO:64.

In at least one embodiment, the 186 kDa protein sequence includes valine(V, Val) at the position corresponding to position 1092 of SEQ ID NO:64.

In at least one embodiment, the 186 kDa protein sequence includeshistidine (H, His) at the position corresponding to position 1637 of SEQID NO:64 and valine (V, Val) at the position corresponding to position1092 of SEQ ID NO:64.

In at least one embodiment, the 186 kDa protein sequence includes atleast five residues selected from:

serine (S, Ser) at the position corresponding to position 86 of SEQ IDNO:64;

glycine (G, Gly) at the position corresponding to position 480 of SEQ IDNO:64;

phenylalanine (F, Phe) at the position corresponding to position 534 ofSEQ ID NO:64;

valine (V, Val) at the position corresponding to position 1124 of SEQ IDNO:64;

aspartic acid (D, Asp) at the position corresponding to position 1157 ofSEQ ID NO:64;

leucine (L, Leu) at the position corresponding to position 1362 of SEQID NO:64; and

serine (S, Ser) at the position corresponding to position 1397 of SEQ IDNO:64.

In at least one such embodiment, the 186 kDa protein sequence furtherincludes one or both of:

-   -   valine (V, Val) at the position corresponding to position 1092        of SEQ ID NO:64; and    -   histidine (H, His) at the position corresponding to position        1637 of SEQ ID NO:64.

In at least one embodiment, the 186 kDa protein sequence includes atleast six residues selected from:

serine (S, Ser) at the position corresponding to position 86 of SEQ IDNO:64;

glycine (G, Gly) at the position corresponding to position 480 of SEQ IDNO:64;

phenylalanine (F, Phe) at the position corresponding to position 534 ofSEQ ID NO:64;

valine (V, Val) at the position corresponding to position 1124 of SEQ IDNO:64;

aspartic acid (D, Asp) at the position corresponding to position 1157 ofSEQ ID NO:64;

leucine (L, Leu) at the position corresponding to position 1362 of SEQID NO:64; and

serine (S, Ser) at the position corresponding to position 1397 of SEQ IDNO:64.

In at least one such embodiment, the 186 kDa protein sequence furtherincludes one or both of:

-   -   valine (V, Val) at the position corresponding to position 1092        of SEQ ID NO:64; and    -   histidine (H, His) at the position corresponding to position        1637 of SEQ ID NO:64.

In at least one embodiment, the 186 kDa protein sequence includes theresidues:

serine (S, Ser) at the position corresponding to position 86 of SEQ IDNO:64;

glycine (G, Gly) at the position corresponding to position 480 of SEQ IDNO:64;

phenylalanine (F, Phe) at the position corresponding to position 534 ofSEQ ID NO:64;

valine (V, Val) at the position corresponding to position 1124 of SEQ IDNO:64;

aspartic acid (D, Asp) at the position corresponding to position 1157 ofSEQ ID NO:64;

leucine (L, Leu) at the position corresponding to position 1362 of SEQID NO:64; and

serine (S, Ser) at the position corresponding to position 1397 of SEQ IDNO:64.

In at least one such embodiment, the 186 kDa protein sequence furtherincludes one or both of:

-   -   valine (V, Val) at the position corresponding to position 1092        of SEQ ID NO:64; and    -   histidine (H, His) at the position corresponding to position        1637 of SEQ ID NO:64.

In at least one embodiment, the sequence of the 186 kDa protein isselected from SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:55,SEQ ID NO:59, SEQ ID NO:62 and variants thereof which include histidine(H, His) at the position corresponding to position 1637 of SEQ ID NO:64.

In at least one embodiment, the sequence of the 186 kDa protein isselected from SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:55,SEQ ID NO:59, SEQ ID NO:62 and variants thereof which include valine (V,Val) at the position corresponding to position 1092 of SEQ ID NO:64.

In at least one embodiment, the sequence of the 186 kDa protein isselected from SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:55,SEQ ID NO:59, SEQ ID NO:62 and variants thereof which include histidine(H, His) at the position corresponding to position 1637 of SEQ ID NO:64and valine (V, Val) at the position corresponding to position 1092 ofSEQ ID NO:64.

In at least one embodiment, the sequence of the 186 kDa protein isselected from SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:55,SEQ ID NO:59, SEQ ID NO:62 and variants thereof which include at leastfive residues selected from:

serine (S, Ser) at the position corresponding to position 86 of SEQ IDNO:64;

glycine (G, Gly) at the position corresponding to position 480 of SEQ IDNO:64;

phenylalanine (F, Phe) at the position corresponding to position 534 ofSEQ ID NO:64;

valine (V, Val) at the position corresponding to position 1124 of SEQ IDNO:64;

aspartic acid (D, Asp) at the position corresponding to position 1157 ofSEQ ID NO:64;

leucine (L, Leu) at the position corresponding to position 1362 of SEQID NO:64; and

serine (S, Ser) at the position corresponding to position 1397 of SEQ IDNO:64.

In at least one embodiment, the variants of the 186 kDa protein sequencefurther include one or both of:

-   -   valine (V, Val) at the position corresponding to position 1092        of SEQ ID NO:64; and    -   histidine (H, His) at the position corresponding to position        1637 of SEQ ID NO:64.

In at least one embodiment, the sequence of the 186 kDa protein isselected from SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:55,SEQ ID NO:59, SEQ ID NO:62 and variants thereof which include at leastsix residues selected from:

serine (S, Ser) at the position corresponding to position 86 of SEQ IDNO:64;

glycine (G, Gly) at the position corresponding to position 480 of SEQ IDNO:64;

phenylalanine (F, Phe) at the position corresponding to position 534 ofSEQ ID NO:64;

valine (V, Val) at the position corresponding to position 1124 of SEQ IDNO:64;

aspartic acid (D, Asp) at the position corresponding to position 1157 ofSEQ ID NO:64;

leucine (L, Leu) at the position corresponding to position 1362 of SEQID NO:64; and

serine (S, Ser) at the position corresponding to position 1397 of SEQ IDNO:64.

In at least one embodiment, the variants of the 186 kDa protein sequencefurther include one or both of:

-   -   valine (V, Val) at the position corresponding to position 1092        of SEQ ID NO:64; and    -   histidine (H, His) at the position corresponding to position        1637 of SEQ ID NO:64.

In at least one embodiment, the sequence of the 186 kDa protein isselected from SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:55,SEQ ID NO:59, SEQ ID NO:62 and variants thereof which include theresidues:

serine (S, Ser) at the position corresponding to position 86 of SEQ IDNO:64;

glycine (G, Gly) at the position corresponding to position 480 of SEQ IDNO:64;

phenylalanine (F, Phe) at the position corresponding to position 534 ofSEQ ID NO:64;

valine (V, Val) at the position corresponding to position 1124 of SEQ IDNO:64;

aspartic acid (D, Asp) at the position corresponding to position 1157 ofSEQ ID NO:64;

leucine (L, Leu) at the position corresponding to position 1362 of SEQID NO:64; and

serine (S, Ser) at the position corresponding to position 1397 of SEQ IDNO:64.

In at least one embodiment, the variants of the 186 kDa protein sequencefurther include one or both of:

-   -   valine (V, Val) at the position corresponding to position 1092        of SEQ ID NO:64; and    -   histidine (H, His) at the position corresponding to position        1637 of SEQ ID NO:64.

Because the 186 kDa protein is encoded by an open reading frame in theCGMMV genome which includes the open reading frame for the 129 kDaprotein, it will be clear to the person of skill in the art that apolyribonucleotide or a polydeoxyribonucleotide as described hereinwhich encodes a 186 kDa protein as described herein will also encode a129 kDa protein as described herein.

In at least one embodiment, the coat protein has an amino acid sequencecomprising valine (V, Val) at the position corresponding to position 156of SEQ ID NO:65. In at least one embodiment, the sequence of the coatprotein is selected from SEQ ID NO:32 and variants thereof comprisingvaline (V, Val) at the position corresponding to position 156 of SEQ IDNO:65.

As will be understood in the art, the degeneracy of the genetic codeallows for some amino acids to be encoded by more than one codon orgroup of three nucleoside residues. Therefore, it is contemplated thatthe sequences of the present polyribonucleotide orpolydeoxyribonucleotide can also include mutations other than thosespecifically described herein such that the polyribonucleotide orpolydeoxyribonucleotide will encode proteins having amino acid sequencesas described herein.

In at least one embodiment, the polydeoxyribonucleotide is configuredfor expression in a host cell so as to permit expression of viralproteins and/or assembly and/or replication of infectious virus in thehost cell, as will be understood by those skilled in the art, who willbe capable of configuring the polydeoxyribonucleotide for expression insuch a host cell without undue experimentation in light of the teachingherein. In at least one embodiment, the host cell is a microorganism. Inat least one embodiment, the host cell is a plant cell.

In another aspect, the present application provides a vector comprisinga polydeoxyribonucleotide as described herein. In at least oneembodiment, the vector is configured for use to genetically modify acell. Thus, a further aspect of the present application provides agenetically modified cell comprising a polydeoxyribonucleotide asdescribed herein. In at least one embodiment, the cell is amicroorganism. In at least one embodiment, the cell is a plant cell.Those skilled in the art would be aware of methods for preparing suchvectors and using them to genetically modify such cells.

Another aspect of the present application provides a composition forpreventing symptoms associated with infection by wild-type CGMMV in aplant, where the composition comprises an attenuated strain of CGMMV ora genetically modified cell as described herein and an agriculturallyacceptable carrier. In at least one embodiment, the compositioncomprises two or more attenuated strains of CGMMV or geneticallymodified cells as described herein.

As used herein, the term “carrier” is intended to refer to a diluent,adjuvant, excipient, or vehicle with which an attenuated strain of CGMMVor a genetically modified cell can be applied or administered to a plantor crop. As used herein, the term “agriculturally acceptable” isintended to refer to carriers and compositions containing such carriersthat are tolerable and do not typically produce untoward reactions to aplant or crop being treated with such carriers and compositions, or to aworker applying such carriers and compositions to a plant or crop undernormal agricultural conditions. Preferably, as used herein, the term“agriculturally acceptable” means approved by a regulatory agency of thefederal or a state government for use in agricultural applications. Suchagriculturally acceptable carriers are well known in the art.

In an additional aspect, the present application provides a compositionfor increasing resistance of a plant to infection by wild-type CGMMV,where the composition comprises an attenuated strain of CGMMV or agenetically modified cell as described herein and an agriculturallyacceptable carrier. In at least one embodiment, the compositioncomprises two or more attenuated strains of CGMMV or geneticallymodified cells as described herein.

Another aspect of the present application provides a method forpreventing symptoms associated with infection by wild-type CGMMV in aplant, where the method includes inoculating the plant with anattenuated strain of CGMMV or with a genetically modified cell asdescribed herein. Methods of inoculating plants with viruses, includingbut not limited to attenuated strains thereof, and/or with cells,including but not limited to microorganisms, genetically modified toexpress such viruses or associated viral proteins, and/or withcompositions thereof are well known in the art, and well within thecapability of the skilled person in light of the teaching of the presentapplication.

A further aspect of the present application provides a method forincreasing resistance of a plant to infection by wild-type CGMMV, wherethe method includes inoculating the plant with an attenuated strain ofCGMMV or with a genetically modified cell as described herein.

In a further aspect, the present application provides a geneticallymodified plant comprising a genome which comprises apolydeoxyribonucleotide as described herein. It is contemplated thatsuch a genetically modified plant may have increased resistance toinfection by wild type strains of CGMMV. In at least one embodiment, theplant is a cucurbit. In at least one embodiment, the plant is a cucumberplant.

As used herein, the terms “about” or “approximately” as applied to anumerical value or range of values are intended to mean that the recitedvalues can vary within an acceptable degree of error for the quantitymeasured given the nature or precision of the measurements, such thatthe variation is considered in the art as equivalent to the recitedvalues and provides the same function or result. For example, the degreeof error can be indicated by the number of significant figures providedfor the measurement, as is understood in the art, and includes but isnot limited to a variation of ±1 in the most precise significant figurereported for the measurement. Typical exemplary degrees of error arewithin 20 percent (%), preferably within 10%, and more preferably within5% of a given value or range of values. Alternatively, and particularlyin biological systems, the terms “about” and “approximately” can meanvalues that are within an order of magnitude, preferably within 5-foldand more preferably within 2-fold of a given value. Numerical quantitiesgiven herein are approximate unless stated otherwise, meaning that theterm “about” or “approximately” can be inferred when not expresslystated.

As used herein, the term “substantially” refers to the complete ornearly complete extent or degree of an action, characteristic, property,state, structure, item, or result. For example, an object that is“substantially” in a given position including but not limited tovertical, horizontal, or adjacent to or aligned with another object,would mean that the object is either completely in that position ornearly completely in that position. The exact allowable degree ofdeviation from absolute completeness may in some cases depend on thespecific context. However, generally speaking, the nearness ofcompletion will be so as to have the same overall result as if absoluteand total completion were obtained.

The use of “substantially” is equally applicable when used in a negativeconnotation to refer to the complete or near complete lack of an action,characteristic, property, state, structure, item, or result. Forexample, a composition that is “substantially free of” an ingredient orelement would either completely lack that ingredient or element, or sonearly completely lack that ingredient or element that the effect wouldbe the same as if it completely lacked that ingredient or element. Inother words, a composition that is “substantially free of” an ingredientor element may still actually contain such item as long as there is nomeasurable or significant effect thereof.

EXAMPLES

Other features of the present invention will become apparent from thefollowing non-limiting examples which illustrate, by way of example, theprinciples of the invention.

Example 1: Cucumber Green Mottle Mosaic Virus (CGMMV) Ontario Strain

Isolation, Cloning and Sequencing of a Wild-Type Ontario Strain

Cucumber green mottle mosaic virus (CGMMV) Ontario strain (also referredto herein as the “wild-type” strain) was extracted from cucumber plantsshowing green mottle and mosaic symptoms collected from a commercialgreenhouse in Ontario. cDNA was synthesized from the CGMMV RNA genomeusing Agilent AccuScrip™ High-Fidelity Reverse Transcriptase (Agilent)and an oligonucleotide primer having the sequenceCGGCTCGAGCCCGTTTCGTCCTTTAGGGACTCGTCAGTGTACTGA-TATAAGTACAGACTGGGCCCCTACCCGGGGAAAGGGGGGATT(SEQ ID NO:1). The full-length genome of CGMMV Ontario strain wasamplified from cDNA by polymerase chain reaction (PCR) using Q5™High-fidelity 2× Master Mix (New England Biolabs) and the primersGTTTTAATTTTAAAATTAAACAAACAACAACAACAACAACAAAC (SEQ ID NO:2) andCCCCGGCTCGAGCCCGTTTCGTCCTTTAGGGACTCGT (SEQ ID NO:3). The PCR product wasdigested with XhoI and cloned into a binary vector pKW8 between the StuIand XhoI sites in 10-beta Escherichia coli (New England Biolabs) byelectroporation. The whole cDNA genome was sequenced using primers whichwere designed based on analysis of genome sequences published in GenBankfor other CGMMV strains. The sequences of the primers are listed inTable 1 below.

TABLE 1 Primers used to sequence the cDNA genome of CGMMV wild-typeOntario strain Primer sequence (5′ to 3′) Sequence identifierCGTACCTCCTGAATGCATCTATC SEQ ID NO: 4 CGGTAACTATACGCAGCACTT SEQ ID NO: 5CAGTTTAGGGTCGATGGTGATG SEQ ID NO: 6 CAAGGCCTTAGTGTGGAAGAA SEQ ID NO: 7CTCGCTTCCGGTGATGATTT SEQ ID NO: 8 GTCGGAACCTCGATGACTTTAC SEQ ID NO: 9GTGACGATACCACTCGCATAAT SEQ ID NO: 10 CTGATGGTCCATACGGGATTAC SEQ ID NO:11 GGCCTTAACTAGGCACACTAAG SEQ ID NO: 12 CCGGGTCTTCTTGAGAATCTTG SEQ IDNO: 13 TGGCTTGGATGTGGTCTATG SEQ ID NO: 14 GGTCGATAAGTTGCTCCCTAAC SEQ IDNO: 15 TAGTCGAGTCTGTCGTCTCTTC SEQ ID NO: 16 CCTGTGTTGAGGCCTATCTTC SEQ IDNO: 17

The full-length cDNA genome sequence of CGMMV Ontario strain is shownbelow.

(SEQ ID NO: 18) GTTTTAATTTTTAAAATTAAACAAACAACAACAACAACAACAAACAATTTAAAACAACAATGGCAAACATTAATGAACAAATCAACAACCAACGCGACGCCGCGGCCAGCGGGAGAAACAATCTCGTTAGCCAATTGGCGTCAAAAAGGGTGTATGACGAGGCTGTTCGCTCGTTGGATCATCAAGACAGACGCCCAAAAATGAACTTTTCTCGTGTGGTCAGCACAGAGCACACCAGGCTTGTAACTGATGCGTATCCGGAGTTTTCGATTAGCTTTACCGCCACCAAGAACTCTGTACACTCCCTTGCGGGTGGTCTGAGGCTCCTTGAACTGGAATATATGATGATGCAAGTGCCCTACGGCTCACCTTGTTATGATATCGGCGGTAACTATACGCAGCACTTGTTCAAAGGTAGATCATATGTGCATTGCTGCAATCCGTGCCTGGATCTTAAAGATGTTGCGAGGAACGTGATGTATAACGATATGGTCACACAACATGTACAGAGGCACAAGGGATCTGGCGGGTGCAGACCTCTTCCAACTTTTCAGATAGATGCATTCAGGAGGTACGATAATTCTCCCTGTGCGGTCACCTGTTCAGACGTTTTCCAAGAGTGTTCCTATGATTTTGGGAGCGGTAGGGATAATCATGCAGTCTCGCTGCATTCAATCTACGATATCCCTTATTCTTCGATCGGACCTGCTCTTCATAGGAAGAACGTGCGAGTTTGTTATGCAGCCTTTCACTTCTCGGAGGCATTGCTTTTAGGTTCACCTGTAGGTAATTTAAATAGTATTGGGGCTCAGTTTAGGGTCGATGGTGATGATGTGCATTTTCTTTTTAGTGAAGAGTCTACTTTGCATTATACTCATAGTTTAGAAAATATCAAATTAATTGTGATGCGTACTTATTTTCCTGCTGATGATAGGTACGTGTATATTAAGGAGTTTATGGTCAAGCGTGTGGATACTTTCTTCTTTAGGTTGGTCAGAGCAGACACACATATGCTTCATAAATCTGTGGGGCACTATTCAAAATCGAAATCTGAGTACTTTGCGCTGAATACCCCTCCGATCTTCCAAGACAAAGCCACGTTTTCTGTGTGGTTTCCTGAGGCGAAGCGTAAGGTGTTGATACCCAAGTTTGAACTTTCAAGATTCCTTTCTGGGAATGTGAAAATCTCTAGGATGCTTGTCGATGCTGATTTCGTCCATACCATTATTAATCACATTAGCACGTATGATAATAAGGCCTTAGTGTGGAAGAATGTTCAGTCCTTTGTGGAATCTATACGCTCAAGAGTAATTGTAAACGGAGTTTCGGTGAAATCTGAATGGAACGTACCGGTTGATCAGCTCACTGATATCTCGTTCTCGATATTCCTTCTCGTGAAGGTTAGGAAGGTACAGATCGAGTTAATGTCTGATAAAGTTGTAATCGAGGCGAGGGGCTTGCTCCGGAGGTTCGCAGACAGTCTTAAATCCGCCGTAGAAGGACTAGGTGATTGCGTCTATGATGCTCTAGTTCAAACCGGCTGGTTTGATACCTCTAGCGACGAACTGAAAGTTTTGCTACCTGAACCGTTTATGACCTTTTCGGATTATCTTGAAGGGATGTACGAGGCAGATGCAAAGATCGAGAGAGAGAGTGTCTCTGAGTTGCTCGCTTCCGGTGACGATTTGTTCAAGAAAATCGATGAGATAAGAAACAATTACAGTGGAGTCGAATTTGATGTAGAGAAATTCCAGGAATTTTGCAAGGAACTGAATGTTAATCCTATGCTAATTGGCCATGTTATCGAAGCTATTTTTTCGCAGAAAGCTGGGGTGACAGTAACGGGTCTGGGTACCCTCTCTCCTGAGATGGGTGCTTCTGTTGCGTTATCCAATACCTCTGTAGATACATGTGAAGATATGGATGTAACTGAAGATATGGAGGATATAGTGTTGATGGCGGACAAGAGTCATTCTTACATGTCCCCAGAAATGGCGAGATGGGCTGATGTAAAATACGACAACAATAAAGGGGGCCTGGTCGAATACAAAGTCGGAACCTCGATGACTTTACCTGCCACCTGGGCAGAGAAGGGTAAGGCTGTCTTACCGTTGTCGGGGATCTGTGTGAGGAAACCCCAATTTTCGAAGCCGCTTGATGAGGAAGACGACTTGAGGTTATCAAACATGAATTTCTTTAAGGTGAGCGATCTGAAGTTGAAGAAAACTATCACTCCAGTTGTTTACACTGGGACCATTCGAGAGAGGCAAATGAAGAATTATATTGATTACTTATCGGCCTCTCTTGGTTCTACGCTGGGTAATCTGGAGAGAATTGTGCGGAGTGATTGGAACGGTACCGAGGAGAGTATGCAAACGTTCGGGTTGTATGACTGCGAAAAGTGCAAGTGGTTACTGTTACCAGCCGAAAAGAAGCACGCATGGGCTGTGGTTCTGGCAAGTGATGATACCACTCGCATAATCTTCCTCTCATATGACGAATCTGGTTCTCCCATAATTGATAAGAGAAACTGGAAGCGATTTGCTGTTTGCTCTGAGACCAAAGTCTATAGCGTAATTCGTAGTTTAGAGGTACTAAATAAGGAAGCAATAGTCGACCCCGGGGTTCATATAACATTAGTTGACGGAGTGCCGGGTTGTGGAAAGACCGCCGAAATTATAGCGAGGGTCAATTGGAAAACCGATCTAGTATTGACTCCCGGGAGGGAGGCGGCTGCTATGATTAGGCGGAGGGCCTGCGCCCTGCACAAGTCACCTGTGGCAACCAGTGACAACGTTAGAACTTTCGATTCTTTTGTGATGAATAAGAAAATCTTCAAGTTTGACGCTGTCTATGTTGACGAGGGTCTGATGGTCCATACGGGTTTACTTAATTTTGCGTTGAAGATCTCAGGTTGTAAAAAGGCCTTCGTCTTTGGTGATGCTAAGCAAATCCCGTTTATAAACAGAGTCATGAATTTTGATTATCCTAAGGAGTTAAGAACTTTAATAGTCGATAATGTAGAGCGTAGGTATGTTACCCATAGGTGTCCTAGAGATGTCACTAGTTTTCTTAATACTATTTACAAAGCCGCTGTCGCTACTACTAGTCCGGTTGTACATTCTGTGAAGGCGATTAAAGTGTCAGGGGCCGGTATTCTGAGGCCCGAGTTGACGAAGATCAAAGGAAAGATAATAACGTTTACTCAATCTGATAAGCAGTCCTTGATCAAGAGTGGGTACAATGACGTGAACACTGTGCATGAAATTCAGGGAGAAACCTTTGAAGAGACGGCGGTTGTGCGTGCCACCCCGACTCCGATAGGTTTAATTGCCCGTGATTCACCACATGTACTAGTGGCCTTAACGAGGCACACTAAGGCAATGGTGTATTATACTGTTGTGTTCGATGCAGTTACAAGTATAATAGCGGATGTGGAAAAGGTCGACCAGTCGATCTTGACTATGTTTGCTACCACTGTGCCTACCAAATAGCAATTAATGCAGAACTCACTGTATGTCCATCGTAATATTTTCCTCCCTGTTAGTAAAACGGGGTTTTATACAGACATGCAGGAGTTCTATGATAGATGCCTTCCTGGGAATTCCTTCGTGCTGAATGATTTCGATGCCGTAACCATGCGGTTGAGGGACAACGAATTTAACCTACAACCTTGTAGGCTAACCTTAAGTAATTTAGATCCAGTACCCGCTTTGGTTAAGAGTGAAGCGCAGAATTTTCTGATTCCCGTTTTGCGTACGGCCTGTGAAAGGCCGCGCATTCCAGGTCTCCTTGAAAATCTTGTAGCTATGATAAAGAGGAATATGAATACTCCTGATCTAGCTGGGACTGTGGATATAACTAATATGTCGATTTCTATAGTAGATAACTTCTTTTCTTCTTTTGTTAGAGACGAGGTTTTGCTTGATCATTTAGATTGTGTTAGGGCTAGTTCCATTCAAAGTTTTTCTGATTGGTTTTCGTGTCAGCCAACCTCGGCGGTTGGTCAATTAGCTAATTTCAATTTCATAGATTTGCCTGCCTTTGATACTTATATGCACATGATTAAGCGGCAGCCCAAGAGTCGGTTGGATACTTCGATTCAGTCTGAATATCCGGCCTTGCAAACTATTGTTTATCACCCTAAAGTGGTAAATGCAGTTTTCGGTCCGGTTTTTAAGTATTTGACCACCAAGTTTCTTAGCATGGTAGATAGTTCTAAGTTTTTCTTTTACACTAGGAAAAAACCAGAAGATCTGCAGGAATTTTTCTCAGATCTCTCTTCCCATTCTGATTATGAGATTCTTGAGCTGGATGTTTCTAAATATGACAAGTCACAATCCGATTTCCATTTCTCTATTGAGATGGCAATTTGGGAAAAATTGGGGCTGGACGATATTTTGGCTTGGATGTGGTCTATGGGTCACAAGAGAACTATACTGCAAGATTTCCAAGCCGGGATAAAGACGCTCATTTACTATCAACGGAAGTCTGGTGATGTAACTACTTTCATAGGTAATACCTTTATTATCGCAGCGTGTGTAGCTAGTATGTTGCCGTTAGACAAGTGTTTTAAAGCTAGTTTTTGTGGTGATGATTCGCTGATCTACCTTCCTAAGGGTTTGGAGTATCCTGATATACAGGCTACTGCCAACTTGGTTTGGAATTTTGAGGCGAAACTTTTCCGAAAGAAGTATGGTTACTTCTGTGGGAAGTATATAATTCACCATGCCAACGGCTGTATTGTTTACCCTGACCCTTTAAAATTAATTAGTAAATTAGGTAATAAGAGTCTTGTAGGGTATGAGCATGTTGAGGAGTTTCGTATATCTCTCCTCGACGTCGCTCATAGTTTGTTTAATGGTGCTTATTTCCATTTACTCGACGATGCAATCCACGAATTATTTCCTAACGCTGGGGGTTGCAGTTTTGTAATTAATTGTTTGTGCAAGTATTTGAGTGATAAGCGCCTTTTCCGTAGTCTTTATATAGATGTCTCTAAGTAAGGTGTCGGTCGAGAACTCATTGAAACCCGAGAAGTTTGTTAAAATCTCTTGGGTCGATAAGTTGCTCCCTAACTATTTTTCCATTCTTAAGTATTTATCTATAACTGACTTTAGCGTAGTTAAAGCTCAGAGCTATGAATCCCTCGTGCCTGTCAAGTTGTTGCGTGGTGTTGATCTTACAAAACACCTTTATGTCACATTGTTGGGCGTTGTGGTTTCTGGTGTATGGAACGTACCGGAATCCTGTAGGGGTGGTGCTACTGTTGCTCTGGTTGACACAAGGATGCATTCTGTTGCAGAGGGAACTATATGCAAATTTTCAGCTCCCGCCACCGTCCGCGAATTCTCTGTTAGGTTCATACCTAACTATTCTGTCGTGGCTGCGGATGCCCTTCGCGATCCTTGGTCTTTATTTGTGAGACTCTCTAATGTAGGGATTAAAGATGGTTTCCATCCTTTGACCTTAGAGGTCGCTTGTTTAGTCGCTACAACTAACTCTATTATCAAAAAGGGTCTTAGAGCTTCTGTAGTCGAGTCTGTCGTCTCTTCCGATCAGTCCATTGTCCTAGATTCTTTATCCGAGAAAGTTGAACCTTTCTTTGATAAAGTTCCTATTTCGGCGGCTGTGATGGCAAGAGACCCCAGTTATAGGTCTAGGTCGCAGTCTGTCGGTGGTCGTGGTAAGCGGCATTCTAAACCTCCAAATCGGAGGTTGGACTCTGCTTCTGAAGAGTCCAGTTCTGTTTCTTTCGAAGATGGCTTACAATCCGATCACACCTAGCAAACTTATTGCGTTTAGTGCTTCTTATGTTCCCGTCAGGACTTTACTTAATTTTCTAGTTGCTTCACAAGGTACCGCCTTCCAGACTCAAGCGGGAAGAGATTCTTTCCGCGAGTCCCTGTCTGCGTTACCCTCGTCTGTCGTAGATATTAATTCTAGGTTCCCAAATGCGGGTTTTTACGCTTTCCTCAACGGTCCTGTGTTGAGGCCTATCTTCGTTTCGCTTCTTAGCTCTACGGATACGCGTAATAGGGTCATTGAGGTTGTAGATCCTAGCAATCCTACGACTGCTGAGTCGCTTAACGCTGTAAAGCGTACTGATGACGCATCTACGGCCGCTAGGGCTGAAATAGATAATTTAATAGAGTCTATTTCTAAGGGTTTTGATGTTTATGATAGGGCTTCATTTGAAGCCGCGTTTTCGGTAGTCTGGTCAGAGGCTACCACCTCGAAAGCTTAGCTTCGAGGGTCTTCTGATGGTGGTGCACACCAAAGTGCATAGTGCTTTCCCGTTCACTTAAATCGAACGGTTTGCTCATTGGTTTGCGGAAACCTCTCACGTGTGGCGTTGAAGTTTCTATGGGCAGTAATTCTGCAAGGGGTTCGAATCCCCCCTTTCCCCGGGTAGGGGCCCA.

The 129 kDa protein encoded by the wild type CGMMV Ontario strain hasthe following sequence.

(SEQ ID NO: 63) MANINEQINNQRDAAASGRNNLVSQLASKRVYDEAVRSLDHQDRRPKMNFSRVVSTEHTRLVTDAYPEFSISFTATKNSVHSLAGGLRLLELEYMMMQVPYGSPCYDIGGNYTQHLFKGRSYVHCCNPCLDLKDVARNVMYNDMVTQHVQRHKGSGGCRPLPTFQIDAFRRYDNSPCAVTCSDVFQECSYDFGSGRDNHAVSLHSIYDIPYSSIGPALHRKNVRVCYAAFHFSEALLLGSPVGNLNSIGAQFRVDGDDVHFLFSEESTLHYTHSLENIKLIVMRTYFPADDRYVYIKEFMVKRVDTFFFRLVRADTHMLHKSVGHYSKSKSEYFALNTPPIFQDKATFSVWFPEAKRKVLIPKFELSRFLSGNVKISRMLVDADFVHTIINHISTYDNKALVWKNVQSFVESIRSRVIVNGVSVKSEWNVPVDQLTDISFSIFLLVKVRKVQIELMSDKVVIEARGLLRRFADSLKSAVEGLGDCVYDALVQTGWFDTSSDELKVLLPEPFMTFSDYLEGMYEADAKIERESVSELLASGDDLFKKIDEIRNNYSGVEFDVEKFQEFCKELNVNPMLIGHVIEAIFSQKAGVTVTGLGTLSPEMGASVALSNTSVDTCEDMDVTEDMEDIVLMADKSHSYMSPEMARWADVKYDNNKGGLVEYKVGTSMTLPATWAEKGKAVLPLSGICVRKPQFSKPLDEEDDLRLSNMNFFKVSDLKLKKTITPVVYTGTIRERQMKNYIDYLSASLGSTLGNLERIVRSDWNGTEESMQTFGLYDCEKCKWLLLPAEKKHAWAVVLASDDTTRIIFLSYDESGSPIIDKRNWKRFAVCSETKVYSVIRSLEVLNKEAIVDPGVHITLVDGVPGCGKTAEIIARVNWKTDLVLTPGREAAAMIRRRACALHKSPVATSDNVRTFDSFVMNKKIFKFDAVYVDEGLMVHTGLLNFALKISGCKKAFVFGDAKQIPFINRVMNFDYPKELRTLIVDNVERRYVTHRCPRDVTSFLNTIYKAAVATTSPVVIISVKAIKVSGAGILRPELTKIKGKIITFTQSDKQSLIKSGYNDVNTVHEIQGETFEETAVVRATPTPIGLIARDSPHVLVALTRHTKAMVYYTVVFDAVTSIIADVEKVDQSILTMFATTVPTK.

The 186 kDa protein encoded by the wild type CGMMV Ontario strain hasthe following sequence.

(SEQ ID NO: 64) MANINEQINNQRDAAASGRNNLVSQLASKRVYDEAVRSLDHQDRRPKMNFSRVVSTEHTRLVTDAYPEFSISFTATKNSVHSLAGGLRLLELEYMMMQVPYGSPCYDIGGNYTQHLFKGRSYVHCCNPCLDLKDVARNVMYNDMVTQHVQRHKGSGGCRPLPTFQIDAFRRYDNSPCAVTCSDVFQECSYDFGSGRDNHAVSLHSIYDIPYSSIGPALHRKNVRVCYAAFHFSEALLLGSPVGNLNSIGAQFRVDGDDVHFLFSEESTLHYTHSLENIKLIVMRTYFPADDRYVYIKEFMVKRVDTFFFRLVRADTHMLHKSVGHYSKSKSEYFALNTPPIFQDKATFSVWFPEAKRKVLIPKFELSRFLSGNVKISRMLVDADFVHTIINHISTYDNKALVWKNVQSFVESIRSRVIVNGVSVKSEWNVPVDQLTDISFSIFLLVKVRKVQIELMSDKVVIEARGLLRRFADSLKSAVEGLGDCVYDALVQTGWFDTSSDELKVLLPEPFMTFSDYLEGMYEADAKIERESVSELLASGDDLFKKIDEIRNNYSGVEFDVEKFQEFCKELNVNPMLIGHVIEAIFSQKAGVTVTGLGTLSPEMGASVALSNTSVDTCEDMDVTEDMEDIVLMADKSHSYMSPEMARWADVKYDNNKGGLVEYKVGTSMTLPATWAEKGKAVLPLSGICVRKPQFSKPLDEEDDLRLSNMNFFKVSDLKLKKTITPVVYTGTIRERQMKNYIDYLSASLGSTLGNLERIVRSDWNGTEESMQTFGLYDCEKCKWLLLPAEKKHAWAVVLASDDTTRIIFLSYDESGSPIIDKRNWKRFAVCSETKVYSVIRSLEVLNKEAIVDPGVHITLVDGVPGCGKTAEIIARVNWKTDLVLTPGREAAAMIRRRACALHKSPVATSDNVRTFDSFVMNKKIFKFDAVYVDEGLMVHTGLLNFALKISGCKKAFVFGDAKQIPFINRVMNFDYPKELRTLIVDNVERRYVTHRCPRDVTSFLNTIYKAAVATTSPVVHSVKAIKVSGAGILRPELTKIKGKIITFTQSDKQSLIKSGYNDVNTVHEIQGETFEETAVVRATPTPIGLIARDSPHVLVALTRHTKAMVYYTVVFDAVTSIIADVEKVDQSILTMFATTVPTKXQLMQNSLYVHRNIFLPVSKTGFYTDMQEFYDRCLPGNSFVLNDFDAVTMRLRDNEFNLQPCRLTLSNLDPVPALVKSEAQNFLIPVLRTACERPRIPGLLENLVAMIKRNMNTPDLAGTVDITNMSISIVDNFFSSFVRDEVLLDHLDCVRASSIQSFSDWFSCQPTSAVGQLANFNFIDLPAFDTYMHMIKRQPKSRLDTSIQSEYPALQTIVYHPKVVNAVFGPVFKYLTTKFLSMVDSSKFFFYTRKKPEDLQEFFSDLSSHSDYEILELDVSKYDKSQSDFHFSIEMAIWEKLGLDDILAWMWSMGHKRTILQDFQAGIKTLIYYQRKSGDVTTFIGNTFIIAACVASMLPLDKCFKASFCGDDSLIYLPKGLEYPDIQATANLVWNFEAKLFRKKYGYFCGKYIIHHANGCIVYPDPLKLISKLGNKSLVGYEHVEEFRISLLDVAHSLFNGAYFHLLDDAIHELFPNAGGCSFVINCLCKYLSDKRLFRSLYIDVSK.

The coat protein encoded by the wild type CGMMV Ontario strain has thefollowing sequence.

(SEQ ID NO: 65) MAYNPITPSKLIAFSASYVPVRTLLNFLVASQGTAFQTQAGRDSFRESLSALPSSVVDINSRFPNAGFYAFLNGPVLRPIFVSLLSSTDTRNRVIEVVDPSNPTTAESLNAVKRTDDASTAARAEIDNLIESISKGFDVYDRASFEAAFS VVWSEATTSKA.

Infection of Cucumber Plants with the Wild-Type Ontario Strain

The CGMMV Ontario strain clone was transformed into Agrobacteriumtumefaciens strain EHA105 by electroporation. The Agrobacteriumtransformants were selected on LB medium plates containing 50 μg/ml ofkanamycin and 20 μg/ml of rifampicin. After confirmation by colony PCR,the Agrobacterium transformants carrying CGMMV Ontario strain werecultured overnight at 30° C. with shaking at 200 rpm in LB medium(lysogeny broth, also known as Luria-Bertani medium) containing 50 μg/mlof kanamycin and 20 μg/ml of rifampicin. The overnight culture was usedto inoculate fresh LB medium containing 50 μg/ml of kanamycin, 20 μg/mlof rifampicin, 10 mM MES (2-(N-morpholino)ethanesulfonic acid), and 200μM acetosyringone (3′,5′-dimethoxy-4′-hydroxyacetophenone) and theculture was incubated with shaking at 30° C. until the optical densityat 600 nm (OD₆₀₀) reached between 0.5 and 1.0.

Bacterial cells were harvested by centrifugation at 4000 g for 10minutes and resuspended in the same volume of Agrobacterium inductionbuffer (10 mM MES, 200 μM acetosyringone). The suspended cells wereincubated at room temperature with gentle shaking (50 rpm) for 3-4hours. The Agrobacterium cells were inoculated into the cotyledon of 1-2week-old cucumber plants by leaf infiltration using a 1 ml needlelesssyringe. After incubation for a further two weeks under laboratorygreenhouse conditions (16 h daylight at 22° C., 8 h darkness at 20° C.),cucumber plant leaves were sampled and tested for the presence of CGMMVby ELISA using a commercial ELISA kit for detecting CGMMV (Agdia) andfollowing the manufacturer's directions. The results shown in Table 2demonstrated that the clone of CGMMV Ontario strain was fullyinfectious.

TABLE 2 ELISA results of cucumber plants inoculated with the CGMMV cloneand wild-type CGMMV isolate after 2 weeks inoculation. Optical Densityat 405 nm Treatment Plant 1 Plant 2 Plant 3 Plant 4 Plant 5 CGMMV Clone1.419 1.401 1.348 1.463 1.378 Wild-type CGMMV 1.609 1.734 1.169 1.4571.357 isolate Negative −0.01 0.003 −0.002 0.002 −0.001

In addition, as seen in FIGS. 1A to C, the CGMMV Ontario strain cloneexpressed in Agrobacterium tumefaciens produced similar symptoms ininfected leaves (FIG. 1A) as those produced by infection with thewild-type CGMMV Ontario strain isolate (FIG. 1B). FIG. 1C showsuninfected leaves as a negative control.

Example 2: Attenuated CGMMV Strains

Mutant CGMMV Ontario Strain ONB

Directed mutation of the cDNA genome of the cloned CGMMV Ontario strain(Example 1) was carried out to introduce mutations (c.1498A>G;c.3430C>T; c.3528A>G; c.4248C>T; and c.6228C>T) corresponding to thoseobserved in the attenuated SH33b strain of CGMMV (Tan et al, Ann.Phytopathol. Soc. Jpn (1997), 63(6): 470-474). These mutations resultedin amino acid substitutions in the encoded viral proteins (E480G andA1124V in the 129 kDa protein; E480G, A1124V, N1157D, and P1397S in the186 kDa protein; and A156V in the coat protein).

Mutations were introduced using the QuikChange™ Lightning MultiSite-Directed Mutagenesis kit (Agilent Technologies), following themanufacturer's instructions, and using the mutagenic primers listed inTable 3. Nucleotide residues indicated in bold indicate sites ofmutation. The resulting mutant CGMMV strain was designated Ontariostrain ONB.

TABLE 3 Primers used to produce mutant CGMMV Ontario strain ONB SequencePrimer sequence (5′ to 3′) IdentifierTCTTAAATCCGCCGTAGGAGGACTAGGTGATTGCG SEQ ID NO: 19CGCAATCACCTAGTCCTCCTACGGCGGATTTAAGA SEQ ID NO: 20TCGATGCAGTTACAAGTATAATAGTGGATGTGGAAAA SEQ ID NO: 21 GGTCGCGACCTTTTCCACATCCACTATTATACTTGTAACTG SEQ ID NO: 22 CATCGACTCACTGTATGTCCATCGTGATATTTTCCTCCCTGTT SEQ ID NO: 23 AGCTAACAGGGAGGAAAATATCACGATGGACATACA SEQ ID NO: 24 GTGAGTCTAAGTTTTTCTTTTACACTAGGAAAAAATCAGAA SEQ ID NO: 25 GATCTGCAGGATCCTGCAGATCTTCTGATTTTTTCCTAGTGTAAAAGA SEQ ID NO: 26 AAAACTTAGAGTAGTCTGGTCAGAGGTTACCACCTCGAAAGCT SEQ ID NO: 27AGCTTTCGAGGTGGTAACCTCTGACCAGACTAC SEQ ID NO: 28

The cDNA genome sequence of CGMMV strain ONB is shown below.

(SEQ ID NO: 29) GTTTTAATTTTTAAAATTAAACAAACAACAACAACAACAACAAACAATTTAAAACAACAATGGCAAACATTAATGAACAAATCAACAACCAACGCGACGCCGCGGCCAGCGGGAGAAACAATCTCGTTAGCCAATTGGCGTCAAAAAGGGTGTATGACGAGGCTGTTCGCTCGTTGGATCATCAAGACAGACGCCCAAAAATGAACTTTTCTCGTGTGGTCAGCACAGAGCACACCAGGCTTGTAACTGATGCGTATCCGGAGTTTTCGATTAGCTTTACCGCCACCAAGAACTCTGTACACTCCCTTGCGGGTGGTCTGAGGCTCCTTGAACTGGAATATATGATGATGCAAGTGCCCTACGGCTCACCTTGTTATGATATCGGCGGTAACTATACGCAGCACTTGTTCAAAGGTAGATCATATGTGCATTGCTGCAATCCGTGCCTGGATCTTAAAGATGTTGCGAGGAACGTGATGTATAACGATATGGTCACACAACATGTACAGAGGCACAAGGGATCTGGCGGGTGCAGACCTCTTCCAACTTTTCAGATAGATGCATTCAGGAGGTACGATAATTCTCCCTGTGCGGTCACCTGTTCAGACGTTTTCCAAGAGTGTTCCTATGATTTTGGGAGCGGTAGGGATAATCATGCAGTCTCGCTGCATTCAATCTACGATATCCCTTATTCTTCGATCGGACCTGCTCTTCATAGGAAGAACGTGCGAGTTTGTTATGCAGCCTTTCACTTCTCGGAGGCATTGCTTTTAGGTTCACCTGTAGGTAATTTAAATAGTATTGGGGCTCAGTTTAGGGTCGATGGTGATGATGTGCATTTTCTTTTTAGTGAAGAGTCTACTTTGCATTATACTCATAGTTTAGAAAATATCAAATTAATTGTGATGCGTACTTATTTTCCTGCTGATGATAGGTACGTGTATATTAAGGAGTTTATGGTCAAGCGTGTGGATACTTTCTTCTTTAGGTTGGTCAGAGCAGACACACATATGCTTCATAAATCTGTGGGGCACTATTCAAAATCGAAATCTGAGTACTTTGCGCTGAATACCCCTCCGATCTTCCAAGACAAAGCCACGTTTTCTGTGTGGTTTCCTGAGGCGAAGCGTAAGGTGTTGATACCCAAGTTTGAACTTTCAAGATTCCTTTCTGGGAATGTGAAAATCTCTAGGATGCTTGTCGATGCTGATTTCGTCCATACCATTATTAATCACATTAGCACGTATGATAATAAGGCCTTAGTGTGGAAGAATGTTCAGTCCTTTGTGGAATCTATACGCTCAAGAGTAATTGTAAACGGAGTTTCGGTGAAATCTGAATGGAACGTACCGGTTGATCAGCTCACTGATATCTCGTTCTCGATATTCCTTCTCGTGAAGGTTAGGAAGGTACAGATCGAGTTAATGTCTGATAAAGTTGTAATCGAGGCGAGGGGCTTGCTCCGGAGGTTCGCAGACAGTCTTAAATCCGCCGTAGGAGGACTAGGTGATTGCGTCTATGATGCTCTAGTTCAAACCGGCTGGTTTGATACCTCTAGCGACGAACTGAAAGTTTTGCTACCTGAACCGTTTATGACCTTTTCGGATTATCTTGAAGGGATGTACGAGGCAGATGCAAAGATCGAGAGAGAGAGTGTCTCTGAGTTGCTCGCTTCCGGTGACGATTTGTTCAAGAAAATCGATGAGATAAGAAACAATTACAGTGGAGTCGAATTTGATGTAGAGAAATTCCAGGAATTTTGCAAGGAACTGAATGTTAATCCTATGCTAATTGGCCATGTTATCGAAGCTATTTTTTCGCAGAAAGCTGGGGTGACAGTAACGGGTCTGGGTACCCTCTCTCCTGAGATGGGTGCTTCTGTTGCGTTATCCAATACCTCTGTAGATACATGTGAAGATATGGATGTAACTGAAGATATGGAGGATATAGTGTTGATGGCGGACAAGAGTCATTCTTACATGTCCCCAGAAATGGCGAGATGGGCTGATGTAAAATACGACAACAATAAAGGGGGCCTGGTCGAATACAAAGTCGGAACCTCGATGACTTTACCTGCCACCTGGGCAGAGAAGGGTAAGGCTGTCTTACCGTTGTCGGGGATCTGTGTGAGGAAACCCCAATTTTCGAAGCCGCTTGATGAGGAAGACGACTTGAGGTTATCAAACATGAATTTCTTTAAGGTGAGCGATCTGAAGTTGAAGAAAACTATCACTCCAGTTGTTTACACTGGGACCATTCGAGAGAGGCAAATGAAGAATTATATTGATTACTTATCGGCCTCTCTTGGTTCTACGCTGGGTAATCTGGAGAGAATTGTGCGGAGTGATTGGAACGGTACCGAGGAGAGTATGCAAACGTTCGGGTTGTATGACTGCGAAAAGTGCAAGTGGTTACTGTTACCAGCCGAAAAGAAGCACGCATGGGCTGTGGTTCTGGCAAGTGATGATACCACTCGCATAATCTTCCTCTCATATGACGAATCTGGTTCTCCCATAATTGATAAGAGAAACTGGAAGCGATTTGCTGTTTGCTCTGAGACCAAAGTCTATAGCGTAATTCGTAGTTTAGAGGTACTAAATAAGGAAGCAATAGTCGACCCCGGGGTTCATATAACATTAGTTGACGGAGTGCCGGGTTGTGGAAAGACCGCCGAAATTATAGCGAGGGTCAATTGGAAAACCGATCTAGTATTGACTCCCGGGAGGGAGGCGGCTGCTATGATTAGGCGGAGGGCCTGCGCCCTGCACAAGTCACCTGTGGCAACCAGTGACAACGTTAGAACTTTCGATTCTTTTGTGATGAATAAGAAAATCTTCAAGTTTGACGCTGTCTATGTTGACGAGGGTCTGATGGTCCATACGGGTTTACTTAATTTTGCGTTGAAGATCTCAGGTTGTAAAAAGGCCTTCGTCTTTGGTGATGCTAAGCAAATCCCGTTTATAAACAGAGTCATGAATTTTGATTATCCTAAGGAGTTAAGAACTTTAATAGTCGATAATGTAGAGCGTAGGTATGTTACCCATAGGTGTCCTAGAGATGTCACTAGTTTTCTTAATACTATTTACAAAGCCGCTGTCGCTACTACTAGTCCGGTTGTACATTCTGTGAAGGCGATTAAAGTGTCAGGGGCCGGTATTCTGAGGCCCGAGTTGACGAAGATCAAAGGAAAGATAATAACGTTTACTCAATCTGATAAGCAGTCCTTGATCAAGAGTGGGTACAATGACGTGAACACTGTGCATGAAATTCAGGGAGAAACCTTTGAAGAGACGGCGGTTGTGCGTGCCACCCCGACTCCGATAGGTTTAATTGCCCGTGATTCACCACATGTACTAGTGGCCTTAACGAGGCACACTAAGGCAATGGTGTATTATACTGTTGTGTTCGATGCAGTTACAAGTATAATAGTGGATGTGGAAAAGGTCGACCAGTCGATCTTGACTATGTTTGCTACCACTGTGCCTACCAAATAGCAATTAATGCAGAACTCACTGTATGTCCATCGTGATATTTTCCTCCCTGTTAGTAAAACGGGGTTTTATACAGACATGCAGGAGTTCTATGATAGATGCCTTCCTGGGAATTCCTTCGTGCTGAATGATTTCGATGCCGTAACCATGCGGTTGAGGGACAACGAATTTAACCTACAACCTTGTAGGCTAACCTTAAGTAATTTAGATCCAGTACCCGCTTTGGTTAAGAGTGAAGCGCAGAATTTTCTGATTCCCGTTTTGCGTACGGCCTGTGAAAGGCCGCGCATTCCAGGTCTCCTTGAAAATCTTGTAGCTATGATAAAGAGGAATATGAATACTCCTGATCTAGCTGGGACTGTGGATATAACTAATATGTCGATTTCTATAGTAGATAACTTCTTTTCTTCTTTTGTTAGAGACGAGGTTTTGCTTGATCATTTAGATTGTGTTAGGGCTAGTTCCATTCAAAGTTTTTCTGATTGGTTTTCGTGTCAGCCAACCTCGGCGGTTGGTCAATTAGCTAATTTCAATTTCATAGATTTGCCTGCCTTTGATACTTATATGCACATGATTAAGCGGCAGCCCAAGAGTCGGTTGGATACTTCGATTCAGTCTGAATATCCGGCCTTGCAAACTATTGTTTATCACCCTAAAGTGGTAAATGCAGTTTTCGGTCCGGTTTTTAAGTATTTGACCACCAAGTTTCTTAGCATGGTAGATAGTTCTAAGTTTTTCTTTTACACTAGGAAAAAATCAGAAGATCTGCAGGAATTTTTCTCAGATCTCTCTTCCCATTCTGATTATGAGATTCTTGAGCTGGATGTTTCTAAATATGACAAGTCACAATCCGATTTCCATTTCTCTATTGAGATGGCAATTTGGGAAAAATTGGGGCTGGACGATATTTTGGCTTGGATGTGGTCTATGGGTCACAAGAGAACTATACTGCAAGATTTCCAAGCCGGGATAAAGACGCTCATTTACTATCAACGGAAGTCTGGTGATGTAACTACTTTCATAGGTAATACCTTTATTATCGCAGCGTGTGTAGCTAGTATGTTGCCGTTAGACAAGTGTTTTAAAGCTAGTTTTTGTGGTGATGATTCGCTGATCTACCTTCCTAAGGGTTTGGAGTATCCTGATATACAGGCTACTGCCAACTTGGTTTGGAATTTTGAGGCGAAACTTTTCCGAAAGAAGTATGGTTACTTCTGTGGGAAGTATATAATTCACCATGCCAACGGCTGTATTGTTTACCCTGACCCTTTAAAATTAATTAGTAAATTAGGTAATAAGAGTCTTGTAGGGTATGAGCATGTTGAGGAGTTTCGTATATCTCTCCTCGACGTCGCTCATAGTTTGTTTAATGGTGCTTATTTCCATTTACTCGACGATGCAATCCACGAATTATTTCCTAACGCTGGGGGTTGCAGTTTTGTAATTAATTGTTTGTGCAAGTATTTGAGTGATAAGCGCCTTTTCCGTAGTCTTTATATAGATGTCTCTAAGTAAGGTGTCGGTCGAGAACTCATTGAAACCCGAGAAGTTTGTTAAAATCTCTTGGGTCGATAAGTTGCTCCCTAACTATTTTTCCATTCTTAAGTATTTATCTATAACTGACTTTAGCGTAGTTAAAGCTCAGAGCTATGAATCCCTCGTGCCTGTCAAGTTGTTGCGTGGTGTTGATCTTACAAAACACCTTTATGTCACATTGTTGGGCGTTGTGGTTTCTGGTGTATGGAACGTACCGGAATCCTGTAGGGGTGGTGCTACTGTTGCTCTGGTTGACACAAGGATGCATTCTGTTGCAGAGGGAACTATATGCAAATTTTCAGCTCCCGCCACCGTCCGCGAATTCTCTGTTAGGTTCATACCTAACTATTCTGTCGTGGCTGCGGATGCCCTTCGCGATCCTTGGTCTTTATTTGTGAGACTCTCTAATGTAGGGATTAAAGATGGTTTCCATCCTTTGACCTTAGAGGTCGCTTGTTTAGTCGCTACAACTAACTCTATTATCAAAAAGGGTCTTAGAGCTTCTGTAGTCGAGTCTGTCGTCTCTTCCGATCAGTCCATTGTCCTAGATTCTTTATCCGAGAAAGTTGAACCTTTCTTTGATAAAGTTCCTATTTCGGCGGCTGTGATGGCAAGAGACCCCAGTTATAGGTCTAGGTCGCAGTCTGTCGGTGGTCGTGGTAAGCGGCATTCTAAACCTCCAAATCGGAGGTTGGACTCTGCTTCTGAAGAGTCCAGTTCTGTTTCTTTCGAAGATGGCTTACAATCCGATCACACCTAGCAAACTTATTGCGTTTAGTGCTTCTTATGTTCCCGTCAGGACTTTACTTAATTTTCTAGTTGCTTCACAAGGTACCGCCTTCCAGACTCAAGCGGGAAGAGATTCTTTCCGCGAGTCCCTGTCTGCGTTACCCTCGTCTGTCGTAGATATTAATTCTAGGTTCCCAAATGCGGGTTTTTACGCTTTCCTCAACGGTCCTGTGTTGAGGCCTATCTTCGTTTCGCTTCTTAGCTCTACGGATACGCGTAATAGGGTCATTGAGGTTGTAGATCCTAGCAATCCTACGACTGCTGAGTCGCTTAACGCTGTAAAGCGTACTGATGACGCATCTACGGCCGCTAGGGCTGAAATAGATAATTTAATAGAGTCTATTTCTAAGGGTTTTGATGTTTATGATAGGGCTTCATTTGAAGCCGCGTTTTCGGTAGTCTGGTCAGAGGTTACCACCTCGAAAGCTTAGCTTCGAGGGTCTTCTGATGGTGGTGCACACCAAAGTGCATAGTGCTTTCCCGTTCACTTAAATCGAACGGTTTGCTCATTGGTTTGCGGAAACCTCTCACGTGTGGCGTTGAAGTTTCTATGGGCAGTAATTCTGCAAGGGGTTCGAATCCCCCCTTTCCCCGGGTAGGGGCCCA.

The 129 kDa protein encoded by CGMMV strain ONB has the followingsequence.

(SEQ ID NO: 30) MANINEQINNQRDAAASGRNNLVSQLASKRVYDEAVRSLDHQDRRPKMNFSRVVSTEHTRLVTDAYPEFSISFTATKNSVHSLAGGLRLLELEYMMMQVPYGSPCYDIGGNYTQHLFKGRSYVHCCNPCLDLKDVARNVMYNDMVTQHVQRHKGSGGCRPLPTFQIDAFRRYDNSPCAVTCSDVFQECSYDFGSGRDNHAVSLHSIYDIPYSSIGPALHRKNVRVCYAAFHFSEALLLGSPVGNLNSIGAQFRVDGDDVHFLFSEESTLHYTHSLENIKLIVMRTYFPADDRYVYIKEFMVKRVDTFFFRLVRADTHMLHKSVGHYSKSKSEYFALNTPPIFQDKATFSVWFPEAKRKVLIPKFELSRFLSGNVKISRMLVDADFVHTIINHISTYDNKALVWKNVQSFVESIRSRVIVNGVSVKSEWNVPVDQLTDISFSIFLLVKVRKVQIELMSDKVVIEARGLLRRFADSLKSAVGGLGDCVYDALVQTGWFDTSSDELKVLLPEPFMTFSDYLEGMYEADAKIERESVSELLASGDDLFKKIDEIRNNYSGVEFDVEKFQEFCKELNVNPMLIGHVIEAIFSQKAGVTVTGLGTLSPEMGASVALSNTSVDTCEDMDVTEDMEDIVLMADKSHSYMSPEMARWADVKYDNNKGGLVEYKVGTSMTLPATWAEKGKAVLPLSGICVRKPQFSKPLDEEDDLRLSNMNFFKVSDLKLKKTITPVVYTGTIRERQMKNYIDYLSASLGSTLGNLERIVRSDWNGTEESMQTFGLYDCEKCKWLLLPAEKKHAWAVVLASDDTTRIIFLSYDESGSPIIDKRNWKRFAVCSETKVYSVIRSLEVLNKEAIVDPGVHITLVDGVPGCGKTAEIIARVNWKTDLVLTPGREAAAMIRRRACALHKSPVATSDNVRTFDSFVMNKKIFKFDAVYVDEGLMVHTGLLNFALKISGCKKAFVFGDAKQIPFINRVMNFDYPKELRTLIVDNVERRYVTHRCPRDVTSFLNTIYKAAVATTSPVVHSVKAIKVSGAGILRPELTKIKGKIITFTQSDKQSLIKSGYNDVNTVHEIQGETFEETAVVRATPTPIGLIARDSPHVLVALTRHTKAMVYYTVVFDAVTSIIVDVEKVDQSILTMFATTVPTK.

The 186 kDa protein encoded by CGMMV strain ONB has the followingsequence.

(SEQ ID NO: 31) MANINEQINNQRDAAASGRNNLVSQLASKRVYDEAVRSLDHQDRRPKMNFSRVVSTEHTRLVTDAYPEFSISFTATKNSVHSLAGGLRLLELEYMMMQVPYGSPCYDIGGNYTQHLFKGRSYVHCCNPCLDLKDVARNVMYNDMVTQHVQRHKGSGGCRPLPTFQIDAFRRYDNSPCAVTCSDVFQECSYDFGSGRDNHAVSLHSIYDIPYSSIGPALHRKNVRVCYAAFHFSEALLLGSPVGNLNSIGAQFRVDGDDVHFLFSEESTLHYTHSLENIKLIVMRTYFPADDRYVYIKEFMVKRVDTFFFRLVRADTHMLHKSVGHYSKSKSEYFALNTPPIFQDKATFSVWFPEAKRKVLIPKFELSRFLSGNVKISRMLVDADFVHTIINHISTYDNKALVWKNVQSFVESIRSRVIVNGVSVKSEWNVPVDQLTDISFSIFLLVKVRKVQIELMSDKVVIEARGLLRRFADSLKSAVGGLGDCVYDALVQTGWFDTSSDELKVLLPEPFMTFSDYLEGMYEADAKIERESVSELLASGDDLFKKIDEIRNNYSGVEFDVEKFQEFCKELNVNPMLIGHVIEAIFSQKAGVTVTGLGTLSPEMGASVALSNTSVDTCEDMDVTEDMEDIVLMADKSHSYMSPEMARWADVKYDNNKGGLVEYKVGTSMTLPATWAEKGKAVLPLSGICVRKPQFSKPLDEEDDLRLSNMNFFKVSDLKLKKTITPVVYTGTIRERQMKNYIDYLSASLGSTLGNLERIVRSDWNGTEESMQTFGLYDCEKCKWLLLPAEKKHAWAVVLASDDTTRIIFLSYDESGSPIIDKRNWKRFAVCSETKVYSVIRSLEVLNKEAIVDPGVHITLVDGVPGCGKTAEIIARVNWKTDLVLTPGREAAAMIRRRACALHKSPVATSDNVRTFDSFVMNKKIFKFDAVYVDEGLMVHTGLLNFALKISGCKKAFVFGDAKQIPFINRVMNFDYPKELRTLIVDNVERRYVTHRCPRDVTSFLNTIYKAAVATTSPVVHSVKAIKVSGAGILRPELTKIKGKIITFTQSDKQSLIKSGYNDVNTVHEIQGETFEETAVVRATPTPIGLIARDSPHVLVALTRHTKAMVYYTVVFDAVTSIIVDVEKVDQSILTMFATTVPTKXQLMQNSLYVHRDIFLPVSKTGFYTDMQEFYDRCLPGNSFVLNDFDAVTMRLRDNEFNLQPCRLTLSNLDPVPALVKSEAQNFLIPVLRTACERPRIPGLLENLVAMIKRNMNTPDLAGTVDITNMSISIVDNFFSSFVRDEVLLDHLDCVRASSIQSFSDWFSCQPTSAVGQLANFNFIDLPAFDTYMHMIKRQPKSRLDTSIQSEYPALQTIVYHPKVVNAVFGPVFKYLTTKFLSMVDSSKFFFYTRKKSEDLQEFFSDLSSHSDYEILELDVSKYDKSQSDFHFSIEMAIWEKLGLDDILAWMWSMGHKRTILQDFQAGIKTLIYYQRKSGDVTTFIGNTFIIAACVASMLPLDKCFKASFCGDDSLIYLPKGLEYPDIQATANLVWNFEAKLFRKKYGYFCGKYIIHHANGCIVYPDPLKLISKLGNKSLVGYEHVEEFRISLLDVAHSLFNGAYFHLLDDAIHELFPNAGGCSFVINCLCKYLSDKRLFRSLYIDVSK.

The coat protein encoded by CGMMV strain ONB has the following sequence.

(SEQ ID NO: 32) MAYNPITPSKLIAFSASYVPVRTLLNFLVASQGTAFQTQAGRDSFRESLSALPSSVVDINSRFPNAGFYAFLNGPVLRPIFVSLLSSTDTRNRVIEVVDPSNPTTAESLNAVKRTDDASTAARAEIDNLIESISKGFDVYDRASFEAAFS VVWSEVTTSKA.

Mutant CGMMV Ontario Strain ONM

Directed mutation of the cDNA genome of the cloned CGMMV Ontario strain(Example 1) was carried out as described above, but using the mutagenicprimers listed in Table 4, to introduce mutations (c.315G>A; c.1660C>T;and c.4144C>T) corresponding to those observed in the attenuatedVIROG-43M strain (Slavokhotova, A. A., et al, American Journal of PlantSciences (2016), 7: 724-732). Nucleotide residues indicated in boldindicate sites of mutation. These mutations resulted in amino acidsubstitutions in the encoded viral proteins (G86S and S534F in the 129kDa protein; and G86S, S534F and P1362L in the 186 kDa protein). Theresulting mutant CGMMV strain was designated Ontario strain ONM.

TABLE 4 Primers used to produce mutant CGMMV Ontario strain ONM SequencePrimer Sequence (5′ to 3′) Identifier CTCCCTTGCGGGTAGTCTGAGGCTCCT SEQ IDNO: 33 AGGAGCCTCAGACTACCCGCAAGGGAG SEQ ID NO: 34AAAGATCGAGAGAGAGAGTGTCTTTGAGTTGCTCGC SEQ ID NO: 35GCGAGCAACTCAAAGACACTCTCTCTCTCGATCTTT SEQ ID NO: 36CTTGCAAACTATTGTTTATCACCTTAAAGTGGTAAA SEQ ID TGCAGTTTTCG NO: 37CGAAAACTGCATTTACCACTTTAAGGTGATAAACAA SEQ ID TAGTTTGCAAG NO: 38

The cDNA genome sequence of CGMMV strain ONM is shown below.

(SEQ ID NO: 39) GTTTTAATTTTTAAAATTAAACAAACAACAACAACAACAACAAACAATTTAAAACAACAATGGCAAACATTAATGAACAAATCAACAACCAACGCGACGCCGCGGCCAGCGGGAGAAACAATCTCGTTAGCCAATTGGCGTCAAAAAGGGTGTATGACGAGGCTGTTCGCTCGTTGGATCATCAAGACAGACGCCCAAAAATGAACTTTTCTCGTGTGGTCAGCACAGAGCACACCAGGCTTGTAACTGATGCGTATCCGGAGTTTTCGATTAGCTTTACCGCCACCAAGAACTCTGTACACTCCCTTGCGGGTAGTCTGAGGCTCCTTGAACTGGAATATATGATGATGCAAGTGCCCTACGGCTCACCTTGTTATGATATCGGCGGTAACTATACGCAGCACTTGTTCAAAGGTAGATCATATGTGCATTGCTGCAATCCGTGCCTGGATCTTAAAGATGTTGCGAGGAACGTGATGTATAACGATATGGTCACACAACATGTACAGAGGCACAAGGGATCTGGCGGGTGCAGACCTCTTCCAACTTTTCAGATAGATGCATTCAGGAGGTACGATAATTCTCCCTGTGCGGTCACCTGTTCAGACGTTTTCCAAGAGTGTTCCTATGATTTTGGGAGCGGTAGGGATAATCATGCAGTCTCGCTGCATTCAATCTACGATATCCCTTATTCTTCGATCGGACCTGCTCTTCATAGGAAGAACGTGCGAGTTTGTTATGCAGCCTTTCACTTCTCGGAGGCATTGCTTTTAGGTTCACCTGTAGGTAATTTAAATAGTATTGGGGCTCAGTTTAGGGTCGATGGTGATGATGTGCATTTTCTTTTTAGTGAAGAGTCTACTTTGCATTATACTCATAGTTTAGAAAATATCAAATTAATTGTGATGCGTACTTATTTTCCTGCTGATGATAGGTACGTGTATATTAAGGAGTTTATGGTCAAGCGTGTGGATACTTTCTTCTTTAGGTTGGTCAGAGCAGACACACATATGCTTCATAAATCTGTGGGGCACTATTCAAAATCGAAATCTGAGTACTTTGCGCTGAATACCCCTCCGATCTTCCAAGACAAAGCCACGTTTTCTGTGTGGTTTCCTGAGGCGAAGCGTAAGGTGTTGATACCCAAGTTTGAACTTTCAAGATTCCTTTCTGGGAATGTGAAAATCTCTAGGATGCTTGTCGATGCTGATTTCGTCCATACCATTATTAATCACATTAGCACGTATGATAATAAGGCCTTAGTGTGGAAGAATGTTCAGTCCTTTGTGGAATCTATACGCTCAAGAGTAATTGTAAACGGAGTTTCGGTGAAATCTGAATGGAACGTACCGGTTGATCAGCTCACTGATATCTCGTTCTCGATATTCCTTCTCGTGAAGGTTAGGAAGGTACAGATCGAGTTAATGTCTGATAAAGTTGTAATCGAGGCGAGGGGCTTGCTCCGGAGGTTCGCAGACAGTCTTAAATCCGCCGTAGAAGGACTAGGTGATTGCGTCTATGATGCTCTAGTTCAAACCGGCTGGTTTGATACCTCTAGCGACGAACTGAAAGTTTTGCTACCTGAACCGTTTATGACCTTTTCGGATTATCTTGAAGGGATGTACGAGGCAGATGCAAAGATCGAGAGAGAGAGTGTCTTTGAGTTGCTCGCTTCCGGTGACGATTTGTTCAAGAAAATCGATGAGATAAGAAACAATTACAGTGGAGTCGAATTTGATGTAGAGAAATTCCAGGAATTTTGCAAGGAACTGAATGTTAATCCTATGCTAATTGGCCATGTTATCGAAGCTATTTTTTCGCAGAAAGCTGGGGTGACAGTAACGGGTCTGGGTACCCTCTCTCCTGAGATGGGTGCTTCTGTTGCGTTATCCAATACCTCTGTAGATACATGTGAAGATATGGATGTAACTGAAGATATGGAGGATATAGTGTTGATGGCGGACAAGAGTCATTCTTACATGTCCCCAGAAATGGCGAGATGGGCTGATGTAAAATACGACAACAATAAAGGGGGCCTGGTCGAATACAAAGTCGGAACCTCGATGACTTTACCTGCCACCTGGGCAGAGAAGGGTAAGGCTGTCTTACCGTTGTCGGGGATCTGTGTGAGGAAACCCCAATTTTCGAAGCCGCTTGATGAGGAAGACGACTTGAGGTTATCAAACATGAATTTCTTTAAGGTGAGCGATCTGAAGTTGAAGAAAACTATCACTCCAGTTGTTTACACTGGGACCATTCGAGAGAGGCAAATGAAGAATTATATTGATTACTTATCGGCCTCTCTTGGTTCTACGCTGGGTAATCTGGAGAGAATTGTGCGGAGTGATTGGAACGGTACCGAGGAGAGTATGCAAACGTTCGGGTTGTATGACTGCGAAAAGTGCAAGTGGTTACTGTTACCAGCCGAAAAGAAGCACGCATGGGCTGTGGTTCTGGCAAGTGATGATACCACTCGCATAATCTTCCTCTCATATGACGAATCTGGTTCTCCCATAATTGATAAGAGAAACTGGAAGCGATTTGCTGTTTGCTCTGAGACCAAAGTCTATAGCGTAATTCGTAGTTTAGAGGTACTAAATAAGGAAGCAATAGTCGACCCCGGGGTTCATATAACATTAGTTGACGGAGTGCCGGGTTGTGGAAAGACCGCCGAAATTATAGCGAGGGTCAATTGGAAAACCGATCTAGTATTGACTCCCGGGAGGGAGGCGGCTGCTATGATTAGGCGGAGGGCCTGCGCCCTGCACAAGTCACCTGTGGCAACCAGTGACAACGTTAGAACTTTCGATTCTTTTGTGATGAATAAGAAAATCTTCAAGTTTGACGCTGTCTATGTTGACGAGGGTCTGATGGTCCATACGGGTTTACTTAATTTTGCGTTGAAGATCTCAGGTTGTAAAAAGGCCTTCGTCTTTGGTGATGCTAAGCAAATCCCGTTTATAAACAGAGTCATGAATTTTGATTATCCTAAGGAGTTAAGAACTTTAATAGTCGATAATGTAGAGCGTAGGTATGTTACCCATAGGTGTCCTAGAGATGTCACTAGTTTTCTTAATACTATTTACAAAGCCGCTGTCGCTACTACTAGTCCGGTTGTACATTCTGTGAAGGCGATTAAAGTGTCAGGGGCCGGTATTCTGAGGCCCGAGTTGACGAAGATCAAAGGAAAGATAATAACGTTTACTCAATCTGATAAGCAGTCCTTGATCAAGAGTGGGTACAATGACGTGAACACTGTGCATGAAATTCAGGGAGAAACCTTTGAAGAGACGGCGGTTGTGCGTGCCACCCCGACTCCGATAGGTTTAATTGCCCGTGATTCACCACATGTACTAGTGGCCTTAACGAGGCACACTAAGGCAATGGTGTATTATACTGTTGTGTTCGATGCAGTTACAAGTATAATAGCGGATGTGGAAAAGGTCGACCAGTCGATCTTGACTATGTTTGCTACCACTGTGCCTACCAAATAGCAATTAATGCAGAACTCACTGTATGTCCATCGTAATATTTTCCTCCCTGTTAGTAAAACGGGGTTTTATACAGACATGCAGGAGTTCTATGATAGATGCCTTCCTGGGAATTCCTTCGTGCTGAATGATTTCGATGCCGTAACCATGCGGTTGAGGGACAACGAATTTAACCTACAACCTTGTAGGCTAACCTTAAGTAATTTAGATCCAGTACCCGCTTTGGTTAAGAGTGAAGCGCAGAATTTTCTGATTCCCGTTTTGCGTACGGCCTGTGAAAGGCCGCGCATTCCAGGTCTCCTTGAAAATCTTGTAGCTATGATAAAGAGGAATATGAATACTCCTGATCTAGCTGGGACTGTGGATATAACTAATATGTCGATTTCTATAGTAGATAACTTCTTTTCTTCTTTTGTTAGAGACGAGGTTTTGCTTGATCATTTAGATTGTGTTAGGGCTAGTTCCATTCAAAGTTTTTCTGATTGGTTTTCGTGTCAGCCAACCTCGGCGGTTGGTCAATTAGCTAATTTCAATTTCATAGATTTGCCTGCCTTTGATACTTATATGCACATGATTAAGCGGCAGCCCAAGAGTCGGTTGGATACTTCGATTCAGTCTGAATATCCGGCCTTGCAAACTATTGTTTATCACCTTAAAGTGGTAAATGCAGTTTTCGGTCCGGTTTTTAAGTATTTGACCACCAAGTTTCTTAGCATGGTAGATAGTTCTAAGTTTTTCTTTTACACTAGGAAAAAACCAGAAGATCTGCAGGAATTTTTCTCAGATCTCTCTTCCCATTCTGATTATGAGATTCTTGAGCTGGATGTTTCTAAATATGACAAGTCACAATCCGATTTCCATTTCTCTATTGAGATGGCAATTTGGGAAAAATTGGGGCTGGACGATATTTTGGCTTGGATGTGGTCTATGGGTCACAAGAGAACTATACTGCAAGATTTCCAAGCCGGGATAAAGACGCTCATTTACTATCAACGGAAGTCTGGTGATGTAACTACTTTCATAGGTAATACCTTTATTATCGCAGCGTGTGTAGCTAGTATGTTGCCGTTAGACAAGTGTTTTAAAGCTAGTTTTTGTGGTGATGATTCGCTGATCTACCTTCCTAAGGGTTTGGAGTATCCTGATATACAGGCTACTGCCAACTTGGTTTGGAATTTTGAGGCGAAACTTTTCCGAAAGAAGTATGGTTACTTCTGTGGGAAGTATATAATTCACCATGCCAACGGCTGTATTGTTTACCCTGACCCTTTAAAATTAATTAGTAAATTAGGTAATAAGAGTCTTGTAGGGTATGAGCATGTTGAGGAGTTTCGTATATCTCTCCTCGACGTCGCTCATAGTTTGTTTAATGGTGCTTATTTCCATTTACTCGACGATGCAATCCACGAATTATTTCCTAACGCTGGGGGTTGCAGTTTTGTAATTAATTGTTTGTGCAAGTATTTGAGTGATAAGCGCCTTTTCCGTAGTCTTTATATAGATGTCTCTAAGTAAGGTGTCGGTCGAGAACTCATTGAAACCCGAGAAGTTTGTTAAAATCTCTTGGGTCGATAAGTTGCTCCCTAACTATTTTTCCATTCTTAAGTATTTATCTATAACTGACTTTAGCGTAGTTAAAGCTCAGAGCTATGAATCCCTCGTGCCTGTCAAGTTGTTGCGTGGTGTTGATCTTACAAAACACCTTTATGTCACATTGTTGGGCGTTGTGGTTTCTGGTGTATGGAACGTACCGGAATCCTGTAGGGGTGGTGCTACTGTTGCTCTGGTTGACACAAGGATGCATTCTGTTGCAGAGGGAACTATATGCAAATTTTCAGCTCCCGCCACCGTCCGCGAATTCTCTGTTAGGTTCATACCTAACTATTCTGTCGTGGCTGCGGATGCCCTTCGCGATCCTTGGTCTTTATTTGTGAGACTCTCTAATGTAGGGATTAAAGATGGTTTCCATCCTTTGACCTTAGAGGTCGCTTGTTTAGTCGCTACAACTAACTCTATTATCAAAAAGGGTCTTAGAGCTTCTGTAGTCGAGTCTGTCGTCTCTTCCGATCAGTCCATTGTCCTAGATTCTTTATCCGAGAAAGTTGAACCTTTCTTTGATAAAGTTCCTATTTCGGCGGCTGTGATGGCAAGAGACCCCAGTTATAGGTCTAGGTCGCAGTCTGTCGGTGGTCGTGGTAAGCGGCATTCTAAACCTCCAAATCGGAGGTTGGACTCTGCTTCTGAAGAGTCCAGTTCTGTTTCTTTCGAAGATGGCTTACAATCCGATCACACCTAGCAAACTTATTGCGTTTAGTGCTTCTTATGTTCCCGTCAGGACTTTACTTAATTTTCTAGTTGCTTCACAAGGTACCGCCTTCCAGACTCAAGCGGGAAGAGATTCTTTCCGCGAGTCCCTGTCTGCGTTACCCTCGTCTGTCGTAGATATTAATTCTAGGTTCCCAAATGCGGGTTTTTACGCTTTCCTCAACGGTCCTGTGTTGAGGCCTATCTTCGTTTCGCTTCTTAGCTCTACGGATACGCGTAATAGGGTCATTGAGGTTGTAGATCCTAGCAATCCTACGACTGCTGAGTCGCTTAACGCTGTAAAGCGTACTGATGACGCATCTACGGCCGCTAGGGCTGAAATAGATAATTTAATAGAGTCTATTTCTAAGGGTTTTGATGTTTATGATAGGGCTTCATTTGAAGCCGCGTTTTCGGTAGTCTGGTCAGAGGCTACCACCTCGAAAGCTTAGCTTCGAGGGTCTTCTGATGGTGGTGCACACCAAAGTGCATAGTGCTTTCCCGTTCACTTAAATCGAACGGTTTGCTCATTGGTTTGCGGAAACCTCTCACGTGTGGCGTTGAAGTTTCTATGGGCAGTAATTCTGCAAGGGGTTCGAATCCCCCCTTTCCCCGGGTAGGGGCCCA.

The 129 kDa protein encoded by CGMMV strain ONM has the followingsequence.

(SEQ ID NO: 40) MANINEQINNQRDAAASGRNNLVSQLASKRVYDEAVRSLDHQDRRPKMNFSRVVSTEHTRLVTDAYPEFSISFTATKNSVHSLAGSLRLLELEYMMMQVPYGSPCYDIGGNYTQHLFKGRSYVHCCNPCLDLKDVARNVMYNDMVTQHVQRHKGSGGCRPLPTFQIDAFRRYDNSPCAVTCSDVFQECSYDFGSGRDNHAVSLHSIYDIPYSSIGPALHRKNVRVCYAAFHFSEALLLGSPVGNLNSIGAQFRVDGDDVHFLFSEESTLHYTHSLENIKLIVMRTYFPADDRYVYIKEFMVKRVDTFFFRLVRADTHMLHKSVGHYSKSKSEYFALNTPPIFQDKATFSVWFPEAKRKVLIPKFELSRFLSGNVKISRMLVDADFVHTIINHISTYDNKALVWKNVQSFVESIRSRVIVNGVSVKSEWNVPVDQLTDISFSIFLLVKVRKVQIELMSDKVVIEARGLLRRFADSLKSAVEGLGDCVYDALVQTGWFDTSSDELKVLLPEPFMTFSDYLEGMYEADAKIERESVFELLASGDDLFKKIDEIRNNYSGVEFDVEKFQEFCKELNVNPMLIGHVIEAIFSQKAGVTVTGLGTLSPEMGASVALSNTSVDTCEDMDVTEDMEDIVLMADKSHSYMSPEMARWADVKYDNNKGGLVEYKVGTSMTLPATWAEKGKAVLPLSGICVRKPQFSKPLDEEDDLRLSNMNFFKVSDLKLKKTITPVVYTGTIRERQMKNYIDYLSASLGSTLGNLERIVRSDWNGTEESMQTFGLYDCEKCKWLLLPAEKKHAWAVVLASDDTTRIIFLSYDESGSPIIDKRNWKRFAVCSETKVYSVIRSLEVLNKEAIVDPGVHITLVDGVPGCGKTAEIIARVNWKTDLVLTPGREAAAMIRRRACALHKSPVATSDNVRTFDSFVMNKKIFKFDAVYVDEGLMVHTGLLNFALKISGCKKAFVFGDAKQIPFINRVMNFDYPKELRTLIVDNVERRYVTHRCPRDVTSFLNTIYKAAVATTSPVVHSVKAIKVSGAGILRPELTKIKGKIITFTQSDKQSLIKSGYNDVNTVHEIQGETFEETAVVRATPTPIGLIARDSPHVLVALTRHTKAMVYYTVVFDAVTSIIADVEKVDQSILTMFATTVPTK.

The 186 kDa protein encoded by CGMMV strain ONM has the followingsequence.

(SEQ ID NO: 41) MANINEQINNQRDAAASGRNNLVSQLASKRVYDEAVRSLDHQDRRPKMNFSRVVSTEHTRLVTDAYPEFSISFTATKNSVHSLAGSLRLLELEYMMMQVPYGSPCYDIGGNYTQHLFKGRSYVHCCNPCLDLKDVARNVMYNDMVTQHVQRHKGSGGCRPLPTFQIDAFRRYDNSPCAVTCSDVFQECSYDFGSGRDNHAVSLHSIYDIPYSSIGPALHRKNVRVCYAAFHFSEALLLGSPVGNLNSIGAQFRVDGDDVHFLFSEESTLHYTHSLENIKLIVMRTYFPADDRYVYIKEFMVKRVDTFFFRLVRADTHMLHKSVGHYSKSKSEYFALNTPPIFQDKATFSVWFPEAKRKVLIPKFELSRFLSGNVKISRMLVDADFVHTIINHISTYDNKALVWKNVQSFVESIRSRVIVNGVSVKSEWNVPVDQLTDISFSIFLLVKVRKVQIELMSDKVVIEARGLLRRFADSLKSAVEGLGDCVYDALVQTGWFDTSSDELKVLLPEPFMTFSDYLEGMYEADAKIERESVFELLASGDDLFKKIDEIRNNYSGVEFDVEKFQEFCKELNVNPMLIGHVIEAIFSQKAGVTVTGLGTLSPEMGASVALSNTSVDTCEDMDVTEDMEDIVLMADKSHSYMSPEMARWADVKYDNNKGGLVEYKVGTSMTLPATWAEKGKAVLPLSGICVRKPQFSKPLDEEDDLRLSNMNFFKVSDLKLKKTITPVVYTGTIRERQMKNYIDYLSASLGSTLGNLERIVRSDWNGTEESMQTFGLYDCEKCKWLLLPAEKKHAWAVVLASDDTTRIIFLSYDESGSPIIDKRNWKRFAVCSETKVYSVIRSLEVLNKEAIVDPGVHITLVDGVPGCGKTAEIIARVNWKTDLVLTPGREAAAMIRRRACALHKSPVATSDNVRTFDSFVMNKKIFKFDAVYVDEGLMVHTGLLNFALKISGCKKAFVFGDAKQIPFINRVMNFDYPKELRTLIVDNVERRYVTHRCPRDVTSFLNTIYKAAVATTSPVVHSVKAIKVSGAGILRPELTKIKGKIITFTQSDKQSLIKSGYNDVNTVHEIQGETFEETAVVRATPTPIGLIARDSPHVLVALTRHTKAMVYYTVVFDAVTSIIADVEKVDQSILTMFATTVPTKXQLMQNSLYVHRNIFLPVSKTGFYTDMQEFYDRCLPGNSFVLNDFDAVTMRLRDNEFNLQPCRLTLSNLDPVPALVKSEAQNFLIPVLRTACERPRIPGLLENLVAMIKRNMNTPDLAGTVDITNMSISIVDNFFSSFVRDEVLLDHLDCVRASSIQSFSDWFSCQPTSAVGQLANFNFIDLPAFDTYMHMIKRQPKSRLDTSIQSEYPALQTIVYHLKVVNAVFGPVFKYLTTKFLSMVDSSKFFFYTRKKPEDLQEFFSDLSSHSDYEILELDVSKYDKSQSDFHFSIEMAIWEKLGLDDILAWMWSMGHKRTILQDFQAGIKTLIYYQRKSGDVTTFIGNTFIIAACVASMLPLDKCFKASFCGDDSLIYLPKGLEYPDIQATANLVWNFEAKLFRKKYGYFCGKYIIHHANGCIVYPDPLKLISKLGNKSLVGYEHVEEFRISLLDVAHSLFNGAYFHLLDDAIHELFPNAGGCSFVINCLCKYLSDKRLFRSLYIDVSK.

The mutant clones ONB and ONM were each transformed into Agrobacteriumtumefaclens strain EHA105 by electroporation and used to inoculate thecotyledon of 1-2 week old cucumber plants under laboratory greenhouseconditions using the method described in Example 1. As seen in FIG. 2B,two weeks after inoculation, mutant strain ONB induced visible symptomsincluding mottle and mosaic symptoms, Likewise, as seen in FIG. 2C, twoweeks after inoculation, mutant strain ONM also induced visiblesymptoms, although these symptoms were milder than those induced by themutant ONB strain. For comparison, FIG. 2A shows a plant exhibitingsymptoms induced by inoculation with the wild-type Ontario strain CGMMVunder the same conditions, and FIG. 2D shows an uninfected control plantgrown under the same conditions.

Mutant CGMMV Ontario Strain ONBM

Directed mutation of the cDNA genome of the cloned CGMMV Ontario strain(Example 1) was carried out as described above to introduce mutationscorresponding to those of mutants ONB and ONM (c.315G>A; c.1498A>G;c.1660C>T; c.3430C>T; c.3528A>G; c.4144C>T; c.4248C>T; and c.6228C>T).These mutations resulted in amino acid substitutions in the encodedviral proteins (G86S, E480G, S534F and A1124V in the 129 kDa protein;G86S, E480G, S534F, A1124V, N1157D, P1362L, and P1397S in the 186 kDaprotein; and A156V in the coat protein). The resulting mutant CGMMVstrain was designated Ontario strain ONBM.

The cDNA genome sequence of CGMMV strain ONBM is shown below.

(SEQ ID NO: 42) GTTTTAATTTTTAAAATTAAACAAACAACAACAACAACAACAAACAATTTAAAACAACAATGGCAAACATTAATGAACAAATCAACAACCAACGCGACGCCGCGGCCAGCGGGAGAAACAATCTCGTTAGCCAATTGGCGTCAAAAAGGGTGTATGACGAGGCTGTTCGCTCGTTGGATCATCAAGACAGACGCCCAAAAATGAACTTTTCTCGTGTGGTCAGCACAGAGCACACCAGGCTTGTAACTGATGCGTATCCGGAGTTTTCGATTAGCTTTACCGCCACCAAGAACTCTGTACACTCCCTTGCGGGTAGTCTGAGGCTCCTTGAACTGGAATATATGATGATGCAAGTGCCCTACGGCTCACCTTGTTATGATATCGGCGGTAACTATACGCAGCACTTGTTCAAAGGTAGATCATATGTGCATTGCTGCAATCCGTGCCTGGATCTTAAAGATGTTGCGAGGAACGTGATGTATAACGATATGGTCACACAACATGTACAGAGGCACAAGGGATCTGGCGGGTGCAGACCTCTTCCAACTTTTCAGATAGATGCATTCAGGAGGTACGATAATTCTCCCTGTGCGGTCACCTGTTCAGACGTTTTCCAAGAGTGTTCCTATGATTTTGGGAGCGGTAGGGATAATCATGCAGTCTCGCTGCATTCAATCTACGATATCCCTTATTCTTCGATCGGACCTGCTCTTCATAGGAAGAACGTGCGAGTTTGTTATGCAGCCTTTCACTTCTCGGAGGCATTGCTTTTAGGTTCACCTGTAGGTAATTTAAATAGTATTGGGGCTCAGTTTAGGGTCGATGGTGATGATGTGCATTTTCTTTTTAGTGAAGAGTCTACTTTGCATTATACTCATAGTTTAGAAAATATCAAATTAATTGTGATGCGTACTTATTTTCCTGCTGATGATAGGTACGTGTATATTAAGGAGTTTATGGTCAAGCGTGTGGATACTTTCTTCTTTAGGTTGGTCAGAGCAGACACACATATGCTTCATAAATCTGTGGGGCACTATTCAAAATCGAAATCTGAGTACTTTGCGCTGAATACCCCTCCGATCTTCCAAGACAAAGCCACGTTTTCTGTGTGGTTTCCTGAGGCGAAGCGTAAGGTGTTGATACCCAAGTTTGAACTTTCAAGATTCCTTTCTGGGAATGTGAAAATCTCTAGGATGCTTGTCGATGCTGATTTCGTCCATACCATTATTAATCACATTAGCACGTATGATAATAAGGCCTTAGTGTGGAAGAATGTTCAGTCCTTTGTGGAATCTATACGCTCAAGAGTAATTGTAAACGGAGTTTCGGTGAAATCTGAATGGAACGTACCGGTTGATCAGCTCACTGATATCTCGTTCTCGATATTCCTTCTCGTGAAGGTTAGGAAGGTACAGATCGAGTTAATGTCTGATAAAGTTGTAATCGAGGCGAGGGGCTTGCTCCGGAGGTTCGCAGACAGTCTTAAATCCGCCGTAGGAGGACTAGGTGATTGCGTCTATGATGCTCTAGTTCAAACCGGCTGGTTTGATACCTCTAGCGACGAACTGAAAGTTTTGCTACCTGAACCGTTTATGACCTTTTCGGATTATCTTGAAGGGATGTACGAGGCAGATGCAAAGATCGAGAGAGAGAGTGTCTTTGAGTTGCTCGCTTCCGGTGACGATTTGTTCAAGAAAATCGATGAGATAAGAAACAATTACAGTGGAGTCGAATTTGATGTAGAGAAATTCCAGGAATTTTGCAAGGAACTGAATGTTAATCCTATGCTAATTGGCCATGTTATCGAAGCTATTTTTTCGCAGAAAGCTGGGGTGACAGTAACGGGTCTGGGTACCCTCTCTCCTGAGATGGGTGCTTCTGTTGCGTTATCCAATACCTCTGTAGATACATGTGAAGATATGGATGTAACTGAAGATATGGAGGATATAGTGTTGATGGCGGACAAGAGTCATTCTTACATGTCCCCAGAAATGGCGAGATGGGCTGATGTAAAATACGACAACAATAAAGGGGGCCTGGTCGAATACAAAGTCGGAACCTCGATGACTTTACCTGCCACCTGGGCAGAGAAGGGTAAGGCTGTCTTACCGTTGTCGGGGATCTGTGTGAGGAAACCCCAATTTTCGAAGCCGCTTGATGAGGAAGACGACTTGAGGTTATCAAACATGAATTTCTTTAAGGTGAGCGATCTGAAGTTGAAGAAAACTATCACTCCAGTTGTTTACACTGGGACCATTCGAGAGAGGCAAATGAAGAATTATATTGATTACTTATCGGCCTCTCTTGGTTCTACGCTGGGTAATCTGGAGAGAATTGTGCGGAGTGATTGGAACGGTACCGAGGAGAGTATGCAAACGTTCGGGTTGTATGACTGCGAAAAGTGCAAGTGGTTACTGTTACCAGCCGAAAAGAAGCACGCATGGGCTGTGGTTCTGGCAAGTGATGATACCACTCGCATAATCTTCCTCTCATATGACGAATCTGGTTCTCCCATAATTGATAAGAGAAACTGGAAGCGATTTGCTGTTTGCTCTGAGACCAAAGTCTATAGCGTAATTCGTAGTTTAGAGGTACTAAATAAGGAAGCAATAGTCGACCCCGGGGTTCATATAACATTAGTTGACGGAGTGCCGGGTTGTGGAAAGACCGCCGAAATTATAGCGAGGGTCAATTGGAAAACCGATCTAGTATTGACTCCCGGGAGGGAGGCGGCTGCTATGATTAGGCGGAGGGCCTGCGCCCTGCACAAGTCACCTGTGGCAACCAGTGACAACGTTAGAACTTTCGATTCTTTTGTGATGAATAAGAAAATCTTCAAGTTTGACGCTGTCTATGTTGACGAGGGTCTGATGGTCCATACGGGTTTACTTAATTTTGCGTTGAAGATCTCAGGTTGTAAAAAGGCCTTCGTCTTTGGTGATGCTAAGCAAATCCCGTTTATAAACAGAGTCATGAATTTTGATTATCCTAAGGAGTTAAGAACTTTAATAGTCGATAATGTAGAGCGTAGGTATGTTACCCATAGGTGTCCTAGAGATGTCACTAGTTTTCTTAATACTATTTACAAAGCCGCTGTCGCTACTACTAGTCCGGTTGTACATTCTGTGAAGGCGATTAAAGTGTCAGGGGCCGGTATTCTGAGGCCCGAGTTGACGAAGATCAAAGGAAAGATAATAACGTTTACTCAATCTGATAAGCAGTCCTTGATCAAGAGTGGGTACAATGACGTGAACACTGTGCATGAAATTCAGGGAGAAACCTTTGAAGAGACGGCGGTTGTGCGTGCCACCCCGACTCCGATAGGTTTAATTGCCCGTGATTCACCACATGTACTAGTGGCCTTAACGAGGCACACTAAGGCAATGGTGTATTATACTGTTGTGTTCGATGCAGTTACAAGTATAATAGTGGATGTGGAAAAGGTCGACCAGTCGATCTTGACTATGTTTGCTACCACTGTGCCTACCAAATAGCAATTAATGCAGAACTCACTGTATGTCCATCGTGATATTTTCCTCCCTGTTAGTAAAACGGGGTTTTATACAGACATGCAGGAGTTCTATGATAGATGCCTTCCTGGGAATTCCTTCGTGCTGAATGATTTCGATGCCGTAACCATGCGGTTGAGGGACAACGAATTTAACCTACAACCTTGTAGGCTAACCTTAAGTAATTTAGATCCAGTACCCGCTTTGGTTAAGAGTGAAGCGCAGAATTTTCTGATTCCCGTTTTGCGTACGGCCTGTGAAAGGCCGCGCATTCCAGGTCTCCTTGAAAATCTTGTAGCTATGATAAAGAGGAATATGAATACTCCTGATCTAGCTGGGACTGTGGATATAACTAATATGTCGATTTCTATAGTAGATAACTTCTTTTCTTCTTTTGTTAGAGACGAGGTTTTGCTTGATCATTTAGATTGTGTTAGGGCTAGTTCCATTCAAAGTTTTTCTGATTGGTTTTCGTGTCAGCCAACCTCGGCGGTTGGTCAATTAGCTAATTTCAATTTCATAGATTTGCCTGCCTTTGATACTTATATGCACATGATTAAGCGGCAGCCCAAGAGTCGGTTGGATACTTCGATTCAGTCTGAATATCCGGCCTTGCAAACTATTGTTTATCACCTTAAAGTGGTAAATGCAGTTTTCGGTCCGGTTTTTAAGTATTTGACCACCAAGTTTCTTAGCATGGTAGATAGTTCTAAGTTTTTCTTTTACACTAGGAAAAAATCAGAAGATCTGCAGGAATTTTTCTCAGATCTCTCTTCCCATTCTGATTATGAGATTCTTGAGCTGGATGTTTCTAAATATGACAAGTCACAATCCGATTTCCATTTCTCTATTGAGATGGCAATTTGGGAAAAATTGGGGCTGGACGATATTTTGGCTTGGATGTGGTCTATGGGTCACAAGAGAACTATACTGCAAGATTTCCAAGCCGGGATAAAGACGCTCATTTACTATCAACGGAAGTCTGGTGATGTAACTACTTTCATAGGTAATACCTTTATTATCGCAGCGTGTGTAGCTAGTATGTTGCCGTTAGACAAGTGTTTTAAAGCTAGTTTTTGTGGTGATGATTCGCTGATCTACCTTCCTAAGGGTTTGGAGTATCCTGATATACAGGCTACTGCCAACTTGGTTTGGAATTTTGAGGCGAAACTTTTCCGAAAGAAGTATGGTTACTTCTGTGGGAAGTATATAATTCACCATGCCAACGGCTGTATTGTTTACCCTGACCCTTTAAAATTAATTAGTAAATTAGGTAATAAGAGTCTTGTAGGGTATGAGCATGTTGAGGAGTTTCGTATATCTCTCCTCGACGTCGCTCATAGTTTGTTTAATGGTGCTTATTTCCATTTACTCGACGATGCAATCCACGAATTATTTCCTAACGCTGGGGGTTGCAGTTTTGTAATTAATTGTTTGTGCAAGTATTTGAGTGATAAGCGCCTTTTCCGTAGTCTTTATATAGATGTCTCTAAGTAAGGTGTCGGTCGAGAACTCATTGAAACCCGAGAAGTTTGTTAAAATCTCTTGGGTCGATAAGTTGCTCCCTAACTATTTTTCCATTCTTAAGTATTTATCTATAACTGACTTTAGCGTAGTTAAAGCTCAGAGCTATGAATCCCTCGTGCCTGTCAAGTTGTTGCGTGGTGTTGATCTTACAAAACACCTTTATGTCACATTGTTGGGCGTTGTGGTTTCTGGTGTATGGAACGTACCGGAATCCTGTAGGGGTGGTGCTACTGTTGCTCTGGTTGACACAAGGATGCATTCTGTTGCAGAGGGAACTATATGCAAATTTTCAGCTCCCGCCACCGTCCGCGAATTCTCTGTTAGGTTCATACCTAACTATTCTGTCGTGGCTGCGGATGCCCTTCGCGATCCTTGGTCTTTATTTGTGAGACTCTCTAATGTAGGGATTAAAGATGGTTTCCATCCTTTGACCTTAGAGGTCGCTTGTTTAGTCGCTACAACTAACTCTATTATCAAAAAGGGTCTTAGAGCTTCTGTAGTCGAGTCTGTCGTCTCTTCCGATCAGTCCATTGTCCTAGATTCTTTATCCGAGAAAGTTGAACCTTTCTTTGATAAAGTTCCTATTTCGGCGGCTGTGATGGCAAGAGACCCCAGTTATAGGTCTAGGTCGCAGTCTGTCGGTGGTCGTGGTAAGCGGCATTCTAAACCTCCAAATCGGAGGTTGGACTCTGCTTCTGAAGAGTCCAGTTCTGTTTCTTTCGAAGATGGCTTACAATCCGATCACACCTAGCAAACTTATTGCGTTTAGTGCTTCTTATGTTCCCGTCAGGACTTTACTTAATTTTCTAGTTGCTTCACAAGGTACCGCCTTCCAGACTCAAGCGGGAAGAGATTCTTTCCGCGAGTCCCTGTCTGCGTTACCCTCGTCTGTCGTAGATATTAATTCTAGGTTCCCAAATGCGGGTTTTTACGCTTTCCTCAACGGTCCTGTGTTGAGGCCTATCTTCGTTTCGCTTCTTAGCTCTACGGATACGCGTAATAGGGTCATTGAGGTTGTAGATCCTAGCAATCCTACGACTGCTGAGTCGCTTAACGCTGTAAAGCGTACTGATGACGCATCTACGGCCGCTAGGGCTGAAATAGATAATTTAATAGAGTCTATTTCTAAGGGTTTTGATGTTTATGATAGGGCTTCATTTGAAGCCGCGTTTTCGGTAGTCTGGTCAGAGGTTACCACCTCGAAAGCTTAGCTTCGAGGGTCTTCTGATGGTGGTGCACACCAAAGTGCATAGTGCTTTCCCGTTCACTTAAATCGAACGGTTTGCTCATTGGTTTGCGGAAACCTCTCACGTGTGGCGTTGAAGTTTCTATGGGCAGTAATTCTGCAAGGGGTTCGAATCCCCCCTTTCCCCGGGTAGGGGCCCA.

The cDNA genome sequence of the attenuated CGMMV strain ONBM differsfrom the cDNA genome sequence of the wild type CGMMV Ontario strain (SEQID NO:18) at least in that:

the nucleotide at position 315 of SEQ ID NO:42 is A;

the nucleotide at position 1498 of SEQ ID NO:42 is G;

the nucleotide at position 1660 of SEQ ID NO:42 is T;

the nucleotide at position 3430 of SEQ ID NO:42 is T;

the nucleotide at position 3528 of SEQ ID NO:42 is G;

the nucleotide at position 4144 of SEQ ID NO:42 is T;

the nucleotide at position 4248 of SEQ ID NO:42 is T; and

the nucleotide at position 6228 of SEQ ID NO:42 is T.

The 129 kDa protein encoded by the attenuated CGMMV strain ONBM has thefollowing sequence.

(SEQ ID NO: 43) MANINEQINNQRDAAASGRNNLVSQLASKRVYDEAVRSLDHQDRRPKMNFSRVVSTEHTRLVTDAYPEFSISFTATKNSVHSLAGSLRLLELEYMMMQVPYGSPCYDIGGNYTQHLFKGRSYVHCCNPCLDLKDVARNVMYNDMVTQHVQRHKGSGGCRPLPTFQIDAFRRYDNSPCAVTCSDVFQECSYDFGSGRDNHAVSLHSIYDIPYSSIGPALHRKNVRVCYAAFHFSEALLLGSPVGNLNSIGAQFRVDGDDVHFLFSEESTLHYTHSLENIKLIVMRTYFPADDRYVYIKEFMVKRVDTFFFRLVRADTHMLHKSVGHYSKSKSEYFALNTPPIFQDKATFSVWFPEAKRKVLIPKFELSRFLSGNVKISRMLVDADFVHTIINHISTYDNKALVWKNVQSFVESIRSRVIVNGVSVKSEWNVPVDQLTDISFSIFLLVKVRKVQIELMSDKVVIEARGLLRRFADSLKSAVGGLGDCVYDALVQTGWFDTSSDELKVLLPEPFMTFSDYLEGMYEADAKIERESVFELLASGDDLFKKIDEIRNNYSGVEFDVEKFQEFCKELNVNPMLIGHVIEAIFSQKAGVTVTGLGTLSPEMGASVALSNTSVDTCEDMDVTEDMEDIVLMADKSHSYMSPEMARWADVKYDNNKGGLVEYKVGTSMTLPATWAEKGKAVLPLSGICVRKPQFSKPLDEEDDLRLSNMNFFKVSDLKLKKTITPVVYTGTIRERQMKNYIDYLSASLGSTLGNLERIVRSDWNGTEESMQTFGLYDCEKCKWLLLPAEKKHAWAVVLASDDTTRIIFLSYDESGSPIIDKRNWKRFAVCSETKVYSVIRSLEVLNKEAIVDPGVHITLVDGVPGCGKTAEIIARVNWKTDLVLTPGREAAAMIRRRACALHKSPVATSDNVRTFDSFVMNKKIFKFDAVYVDEGLMVHTGLLNFALKISGCKKAFVFGDAKQIPFINRVMNFDYPKELRTLIVDNVERRYVTHRCPRDVTSFLNTIYKAAVATTSPVVHSVKAIKVSGAGILRPELTKIKGKIITFTQSDKQSLIKSGYNDVNTVHEIQGETFEETAVVRATPTPIGLIARDSPHVLVALTRHTKAMVYYTVVFDAVTSIIVDVEKVDQSILTMFATTVPTK.

The 129 kDa protein encoded by the attenuated CGMMV strain ONBM differsfrom the 129 kDa protein encoded by the wild type CGMMV Ontario strain(SEQ ID NO:63) at least in that:

position 86 of SEQ ID NO:43 is serine (S, Ser);

position 480 of SEQ ID NO:43 is glycine (G, Gly);

position 534 of SEQ ID NO:43 is phenylalanine (F, Phe); and

position 1124 of SEQ ID NO:43 is valine (V, Val).

The 186 kDa protein encoded by the attenuated CGMMV strain ONBM has thefollowing sequence.

(SEQ ID NO: 44) MANINEQINNQRDAAASGRNNLVSQLASKRVYDEAVRSLDHQDRRPKMNFSRVVSTEHTRLVTDAYPEFSISFTATKNSVHSLAGSLRLLELEYMMMQVPYGSPCYDIGGNYTQHLFKGRSYVHCCNPCLDLKDVARNVMYNDMVTQHVQRHKGSGGCRPLPTFQIDAFRRYDNSPCAVTCSDVFQECSYDFGSGRDNHAVSLHSIYDIPYSSIGPALHRKNVRVCYAAFHFSEALLLGSPVGNLNSIGAQFRVDGDDVHFLFSEESTLHYTHSLENIKLIVMRTYFPADDRYVYIKEFMVKRVDTFFFRLVRADTHMLHKSVGHYSKSKSEYFALNTPPIFQDKATFSVWFPEAKRKVLIPKFELSRFLSGNVKISRMLVDADFVHTIINHISTYDNKALVWKNVQSFVESIRSRVIVNGVSVKSEWNVPVDQLTDISFSIFLLVKVRKVQIELMSDKVVIEARGLLRRFADSLKSAVGGLGDCVYDALVQTGWFDTSSDELKVLLPEPFMTFSDYLEGMYEADAKIERESVFELLASGDDLFKKIDEIRNNYSGVEFDVEKFQEFCKELNVNPMLIGHVIEAIFSQKAGVTVTGLGTLSPEMGASVALSNTSVDTCEDMDVTEDMEDIVLMADKSHSYMSPEMARWADVKYDNNKGGLVEYKVGTSMTLPATWAEKGKAVLPLSGICVRKPQFSKPLDEEDDLRLSNMNFFKVSDLKLKKTITPVVYTGTIRERQMKNYIDYLSASLGSTLGNLERIVRSDWNGTEESMQTFGLYDCEKCKWLLLPAEKKHAWAVVLASDDTTRIIFLSYDESGSPIIDKRNWKRFAVCSETKVYSVIRSLEVLNKEAIVDPGVHITLVDGVPGCGKTAEIIARVNWKTDLVLTPGREAAAMIRRRACALHKSPVATSDNVRTFDSFVMNKKIFKFDAVYVDEGLMVHTGLLNFALKISGCKKAFVFGDAKQIPFINRVMNFDYPKELRTLIVDNVERRYVTHRCPRDVTSFLNTIYKAAVATTSPVVHSVKAIKVSGAGILRPELTKIKGKIITFTQSDKQSLIKSGYNDVNTVHEIQGETFEETAVVRATPTPIGLIARDSPHVLVALTRHTKAMVYYTVVFDAVTSIIVDVEKVDQSILTMFATTVPTKXQLMQNSLYVHRDIFLPVSKTGFYTDMQEFYDRCLPGNSFVLNDFDAVTMRLRDNEFNLQPCRLTLSNLDPVPALVKSEAQNFLIPVLRTACERPRIPGLLENLVAMIKRNMNTPDLAGTVDITNMSISIVDNFFSSFVRDEVLLDHLDCVRASSIQSFSDWFSCQPTSAVGQLANFNFIDLPAFDTYMHMIKRQPKSRLDTSIQSEYPALQTIVYHLKVVNAVFGPVFKYLTTKFLSMVDSSKFFFYTRKKSEDLQEFFSDLSSHSDYEILELDVSKYDKSQSDFHFSIEMAIWEKLGLDDILAWMWSMGHKRTILQDFQAGIKTLIYYQRKSGDVTTFIGNTFIIAACVASMLPLDKCFKASFCGDDSLIYLPKGLEYPDIQATANLVWNFEAKLFRKKYGYFCGKYIIHHANGCIVYPDPLKLISKLGNKSLVGYEHVEEFRISLLDVAHSLFNGAYFHLLDDAIHELFPNAGGCSFVINCLCKYLSDKRLFRSLYIDVSK.

The 186 kDa protein encoded by the attenuated CGMMV strain ONBM differsfrom the 186 kDa protein encoded by the wild type CGMMV Ontario strain(SEQ ID NO:64) at least in that:

position 86 of SEQ ID NO:44 is serine (S, Ser);

position 480 of SEQ ID NO:44 is glycine (G, Gly);

position 534 of SEQ ID NO:44 is phenylalanine (F, Phe);

position 1124 of SEQ ID NO:44 is valine (V, Val);

position 1157 of SEQ ID NO:44 is aspartic acid (D, Asp);

position 1362 of SEQ ID NO:44 is leucine (L, Leu); and

position 1397 of SEQ ID NO:44 is serine (S, Ser).

The coat protein encoded by the attenuated CGMMV strain ONBM has thesequence of SEQ ID NO:32 and differs from the coat protein encoded bythe wild type CGMMV Ontario strain (SEQ ID NO:65) at least in thatposition 156 of SEQ ID NO:32 is valine (V, Val).

Mutant CGMMV Ontario Strain ONBM-2

Directed mutation of the cDNA genome of the cloned CGMMV Ontario strain(Example 1) was carried out as described above to introduce mutationscorresponding to those of mutants ONB and ONM (c.315G>A; c.1498A>G;c.1660C>T; c.3430C>T; c.3528A>G; c.4144C>T; c.4248C>T; and c.6228C>T) inaddition to the mutation c.3334C>T introduced by randomly replacing C byT during PCR amplification. These mutations resulted in amino acidsubstitutions in the encoded viral proteins (G86S, E480G, S534F, A1092Vand A1124V in the 129 kDa protein; G86S, E480G, S534F, A1092V, A1124V,N1157D, P1362L, and P1397S in the 186 kDa protein; and A156V in the coatprotein). The resulting mutant CGMMV strain was designated Ontariostrain ONBM-2.

The cDNA genome sequence of CGMMV strain ONBM-2 is shown below.

(SEQ ID NO: 45) GTTTTAATTTTTAAAATTAAACAAACAACAACAACAACAACAAACAATTTAAAACAACAATGGCAAACATTAATGAACAAATCAACAACCAACGCGACGCCGCGGCCAGCGGGAGAAACAATCTCGTTAGCCAATTGGCGTCAAAAAGGGTGTATGACGAGGCTGTTCGCTCGTTGGATCATCAAGACAGACGCCCAAAAATGAACTTTTCTCGTGTGGTCAGCACAGAGCACACCAGGCTTGTAACTGATGCGTATCCGGAGTTTTCGATTAGCTTTACCGCCACCAAGAACTCTGTACACTCCCTTGCGGGTAGTCTGAGGCTCCTTGAACTGGAATATATGATGATGCAAGTGCCCTACGGCTCACCTTGTTATGATATCGGCGGTAACTATACGCAGCACTTGTTCAAAGGTAGATCATATGTGCATTGCTGCAATCCGTGCCTGGATCTTAAAGATGTTGCGAGGAACGTGATGTATAACGATATGGTCACACAACATGTACAGAGGCACAAGGGATCTGGCGGGTGCAGACCTCTTCCAACTTTTCAGATAGATGCATTCAGGAGGTACGATAATTCTCCCTGTGCGGTCACCTGTTCAGACGTTTTCCAAGAGTGTTCCTATGATTTTGGGAGCGGTAGGGATAATCATGCAGTCTCGCTGCATTCAATCTACGATATCCCTTATTCTTCGATCGGACCTGCTCTTCATAGGAAGAACGTGCGAGTTTGTTATGCAGCCTTTCACTTCTCGGAGGCATTGCTTTTAGGTTCACCTGTAGGTAATTTAAATAGTATTGGGGCTCAGTTTAGGGTCGATGGTGATGATGTGCATTTTCTTTTTAGTGAAGAGTCTACTTTGCATTATACTCATAGTTTAGAAAATATCAAATTAATTGTGATGCGTACTTATTTTCCTGCTGATGATAGGTACGTGTATATTAAGGAGTTTATGGTCAAGCGTGTGGATACTTTCTTCTTTAGGTTGGTCAGAGCAGACACACATATGCTTCATAAATCTGTGGGGCACTATTCAAAATCGAAATCTGAGTACTTTGCGCTGAATACCCCTCCGATCTTCCAAGACAAAGCCACGTTTTCTGTGTGGTTTCCTGAGGCGAAGCGTAAGGTGTTGATACCCAAGTTTGAACTTTCAAGATTCCTTTCTGGGAATGTGAAAATCTCTAGGATGCTTGTCGATGCTGATTTCGTCCATACCATTATTAATCACATTAGCACGTATGATAATAAGGCCTTAGTGTGGAAGAATGTTCAGTCCTTTGTGGAATCTATACGCTCAAGAGTAATTGTAAACGGAGTTTCGGTGAAATCTGAATGGAACGTACCGGTTGATCAGCTCACTGATATCTCGTTCTCGATATTCCTTCTCGTGAAGGTTAGGAAGGTACAGATCGAGTTAATGTCTGATAAAGTTGTAATCGAGGCGAGGGGCTTGCTCCGGAGGTTCGCAGACAGTCTTAAATCCGCCGTAGGAGGACTAGGTGATTGCGTCTATGATGCTCTAGTTCAAACCGGCTGGTTTGATACCTCTAGCGACGAACTGAAAGTTTTGCTACCTGAACCGTTTATGACCTTTTCGGATTATCTTGAAGGGATGTACGAGGCAGATGCAAAGATCGAGAGAGAGAGTGTCTTTGAGTTGCTCGCTTCCGGTGACGATTTGTTCAAGAAAATCGATGAGATAAGAAACAATTACAGTGGAGTCGAATTTGATGTAGAGAAATTCCAGGAATTTTGCAAGGAACTGAATGTTAATCCTATGCTAATTGGCCATGTTATCGAAGCTATTTTTTCGCAGAAAGCTGGGGTGACAGTAACGGGTCTGGGTACCCTCTCTCCTGAGATGGGTGCTTCTGTTGCGTTATCCAATACCTCTGTAGATACATGTGAAGATATGGATGTAACTGAAGATATGGAGGATATAGTGTTGATGGCGGACAAGAGTCATTCTTACATGTCCCCAGAAATGGCGAGATGGGCTGATGTAAAATACGACAACAATAAAGGGGGCCTGGTCGAATACAAAGTCGGAACCTCGATGACTTTACCTGCCACCTGGGCAGAGAAGGGTAAGGCTGTCTTACCGTTGTCGGGGATCTGTGTGAGGAAACCCCAATTTTCGAAGCCGCTTGATGAGGAAGACGACTTGAGGTTATCAAACATGAATTTCTTTAAGGTGAGCGATCTGAAGTTGAAGAAAACTATCACTCCAGTTGTTTACACTGGGACCATTCGAGAGAGGCAAATGAAGAATTATATTGATTACTTATCGGCCTCTCTTGGTTCTACGCTGGGTAATCTGGAGAGAATTGTGCGGAGTGATTGGAACGGTACCGAGGAGAGTATGCAAACGTTCGGGTTGTATGACTGCGAAAAGTGCAAGTGGTTACTGTTACCAGCCGAAAAGAAGCACGCATGGGCTGTGGTTCTGGCAAGTGATGATACCACTCGCATAATCTTCCTCTCATATGACGAATCTGGTTCTCCCATAATTGATAAGAGAAACTGGAAGCGATTTGCTGTTTGCTCTGAGACCAAAGTCTATAGCGTAATTCGTAGTTTAGAGGTACTAAATAAGGAAGCAATAGTCGACCCCGGGGTTCATATAACATTAGTTGACGGAGTGCCGGGTTGTGGAAAGACCGCCGAAATTATAGCGAGGGTCAATTGGAAAACCGATCTAGTATTGACTCCCGGGAGGGAGGCGGCTGCTATGATTAGGCGGAGGGCCTGCGCCCTGCACAAGTCACCTGTGGCAACCAGTGACAACGTTAGAACTTTCGATTCTTTTGTGATGAATAAGAAAATCTTCAAGTTTGACGCTGTCTATGTTGACGAGGGTCTGATGGTCCATACGGGTTTACTTAATTTTGCGTTGAAGATCTCAGGTTGTAAAAAGGCCTTCGTCTTTGGTGATGCTAAGCAAATCCCGTTTATAAACAGAGTCATGAATTTTGATTATCCTAAGGAGTTAAGAACTTTAATAGTCGATAATGTAGAGCGTAGGTATGTTACCCATAGGTGTCCTAGAGATGTCACTAGTTTTCTTAATACTATTTACAAAGCCGCTGTCGCTACTACTAGTCCGGTTGTACATTCTGTGAAGGCGATTAAAGTGTCAGGGGCCGGTATTCTGAGGCCCGAGTTGACGAAGATCAAAGGAAAGATAATAACGTTTACTCAATCTGATAAGCAGTCCTTGATCAAGAGTGGGTACAATGACGTGAACACTGTGCATGAAATTCAGGGAGAAACCTTTGAAGAGACGGCGGTTGTGCGTGCCACCCCGACTCCGATAGGTTTAATTGTCCGTGATTCACCACATGTACTAGTGGCCTTAACGAGGCACACTAAGGCAATGGTGTATTATACTGTTGTGTTCGATGCAGTTACAAGTATAATAGTGGATGTGGAAAAGGTCGACCAGTCGATCTTGACTATGTTTGCTACCACTGTGCCTACCAAATAGCAATTAATGCAGAACTCACTGTATGTCCATCGTGATATTTTCCTCCCTGTTAGTAAAACGGGGTTTTATACAGACATGCAGGAGTTCTATGATAGATGCCTTCCTGGGAATTCCTTCGTGCTGAATGATTTCGATGCCGTAACCATGCGGTTGAGGGACAACGAATTTAACCTACAACCTTGTAGGCTAACCTTAAGTAATTTAGATCCAGTACCCGCTTTGGTTAAGAGTGAAGCGCAGAATTTTCTGATTCCCGTTTTGCGTACGGCCTGTGAAAGGCCGCGCATTCCAGGTCTCCTTGAAAATCTTGTAGCTATGATAAAGAGGAATATGAATACTCCTGATCTAGCTGGGACTGTGGATATAACTAATATGTCGATTTCTATAGTAGATAACTTCTTTTCTTCTTTTGTTAGAGACGAGGTTTTGCTTGATCATTTAGATTGTGTTAGGGCTAGTTCCATTCAAAGTTTTTCTGATTGGTTTTCGTGTCAGCCAACCTCGGCGGTTGGTCAATTAGCTAATTTCAATTTCATAGATTTGCCTGCCTTTGATACTTATATGCACATGATTAAGCGGCAGCCCAAGAGTCGGTTGGATACTTCGATTCAGTCTGAATATCCGGCCTTGCAAACTATTGTTTATCACCTTAAAGTGGTAAATGCAGTTTTCGGTCCGGTTTTTAAGTATTTGACCACCAAGTTTCTTAGCATGGTAGATAGTTCTAAGTTTTTCTTTTACACTAGGAAAAAATCAGAAGATCTGCAGGAATTTTTCTCAGATCTCTCTTCCCATTCTGATTATGAGATTCTTGAGCTGGATGTTTCTAAATATGACAAGTCACAATCCGATTTCCATTTCTCTATTGAGATGGCAATTTGGGAAAAATTGGGGCTGGACGATATTTTGGCTTGGATGTGGTCTATGGGTCACAAGAGAACTATACTGCAAGATTTCCAAGCCGGGATAAAGACGCTCATTTACTATCAACGGAAGTCTGGTGATGTAACTACTTTCATAGGTAATACCTTTATTATCGCAGCGTGTGTAGCTAGTATGTTGCCGTTAGACAAGTGTTTTAAAGCTAGTTTTTGTGGTGATGATTCGCTGATCTACCTTCCTAAGGGTTTGGAGTATCCTGATATACAGGCTACTGCCAACTTGGTTTGGAATTTTGAGGCGAAACTTTTCCGAAAGAAGTATGGTTACTTCTGTGGGAAGTATATAATTCACCATGCCAACGGCTGTATTGTTTACCCTGACCCTTTAAAATTAATTAGTAAATTAGGTAATAAGAGTCTTGTAGGGTATGAGCATGTTGAGGAGTTTCGTATATCTCTCCTCGACGTCGCTCATAGTTTGTTTAATGGTGCTTATTTCCATTTACTCGACGATGCAATCCACGAATTATTTCCTAACGCTGGGGGTTGCAGTTTTGTAATTAATTGTTTGTGCAAGTATTTGAGTGATAAGCGCCTTTTCCGTAGTCTTTATATAGATGTCTCTAAGTAAGGTGTCGGTCGAGAACTCATTGAAACCCGAGAAGTTTGTTAAAATCTCTTGGGTCGATAAGTTGCTCCCTAACTATTTTTCCATTCTTAAGTATTTATCTATAACTGACTTTAGCGTAGTTAAAGCTCAGAGCTATGAATCCCTCGTGCCTGTCAAGTTGTTGCGTGGTGTTGATCTTACAAAACACCTTTATGTCACATTGTTGGGCGTTGTGGTTTCTGGTGTATGGAACGTACCGGAATCCTGTAGGGGTGGTGCTACTGTTGCTCTGGTTGACACAAGGATGCATTCTGTTGCAGAGGGAACTATATGCAAATTTTCAGCTCCCGCCACCGTCCGCGAATTCTCTGTTAGGTTCATACCTAACTATTCTGTCGTGGCTGCGGATGCCCTTCGCGATCCTTGGTCTTTATTTGTGAGACTCTCTAATGTAGGGATTAAAGATGGTTTCCATCCTTTGACCTTAGAGGTCGCTTGTTTAGTCGCTACAACTAACTCTATTATCAAAAAGGGTCTTAGAGCTTCTGTAGTCGAGTCTGTCGTCTCTTCCGATCAGTCCATTGTCCTAGATTCTTTATCCGAGAAAGTTGAACCTTTCTTTGATAAAGTTCCTATTTCGGCGGCTGTGATGGCAAGAGACCCCAGTTATAGGTCTAGGTCGCAGTCTGTCGGTGGTCGTGGTAAGCGGCATTCTAAACCTCCAAATCGGAGGTTGGACTCTGCTTCTGAAGAGTCCAGTTCTGTTTCTTTCGAAGATGGCTTACAATCCGATCACACCTAGCAAACTTATTGCGTTTAGTGCTTCTTATGTTCCCGTCAGGACTTTACTTAATTTTCTAGTTGCTTCACAAGGTACCGCCTTCCAGACTCAAGCGGGAAGAGATTCTTTCCGCGAGTCCCTGTCTGCGTTACCCTCGTCTGTCGTAGATATTAATTCTAGGTTCCCAAATGCGGGTTTTTACGCTTTCCTCAACGGTCCTGTGTTGAGGCCTATCTTCGTTTCGCTTCTTAGCTCTACGGATACGCGTAATAGGGTCATTGAGGTTGTAGATCCTAGCAATCCTACGACTGCTGAGTCGCTTAACGCTGTAAAGCGTACTGATGACGCATCTACGGCCGCTAGGGCTGAAATAGATAATTTAATAGAGTCTATTTCTAAGGGTTTTGATGTTTATGATAGGGCTTCATTTGAAGCCGCGTTTTCGGTAGTCTGGTCAGAGGTTACCACCTCGAAAGCTTAGCTTCGAGGGTCTTCTGATGGTGGTGCACACCAAAGTGCATAGTGCTTTCCCGTTCACTTAAATCGAACGGTTTGCTCATTGGTTTGCGGAAACCTCTCACGTGTGGCGTTGAAGTTTCTATGGGCAGTAATTCTGCAAGGGGTTCGAATCCCCCCTTTCCCCGGGTAGGGGCCCA.

The cDNA genome sequence of the attenuated CGMMV strain ONBM-2 differsfrom the cDNA genome sequence of the wild type CGMMV Ontario strain (SEQID NO: 18) at least in that:

the nucleotide at position 315 of SEQ ID NO:45 is A;

the nucleotide at position 1498 of SEQ ID NO:45 is G;

the nucleotide at position 1660 of SEQ ID NO:45 is T;

the nucleotide at position 3334 of SEQ ID NO:45 is T;

the nucleotide at position 3430 of SEQ ID NO:45 is T;

the nucleotide at position 3528 of SEQ ID NO:45 is G;

the nucleotide at position 4144 of SEQ ID NO:45 is T;

the nucleotide at position 4248 of SEQ ID NO:45 is T; and

the nucleotide at position 6228 of SEQ ID NO:45 is T.

The 129 kDa protein encoded by the attenuated CGMMV strain ONBM-2 hasthe following sequence.

(SEQ ID NO: 46) MANINEQINNQRDAAASGRNNLVSQLASKRVYDEAVRSLDHQDRRPKMNFSRVVSTEHTRLVTDAYPEFSISFTATKNSVHSLAGSLRLLELEYMMMQVPYGSPCYDIGGNYTQHLFKGRSYVHCCNPCLDLKDVARNVMYNDMVTQHVQRHKGSGGCRPLPTFQIDAFRRYDNSPCAVTCSDVFQECSYDFGSGRDNHAVSLHSIYDIPYSSIGPALHRKNVRVCYAAFHFSEALLLGSPVGNLNSIGAQFRVDGDDVIIFLFSEESTLHYTHSLENIKLIVMRTYFPADDRYVYIKEFMVKRVDTFFFRLVRADTHMLHKSVGHYSKSKSEYFALNTPPIFQDKATFSVWFPEAKRKVLIPKFELSRFLSGNVKISRMLVDADFVHTIINHISTYDNKALVWKNVQSFVESIRSRVIVNGVSVKSEWNVPVDQLTDISFSIFLLVKVRKVQIELMSDKVVIEARGLLRRFADSLKSAVGGLGDCVYDALVQTGWFDTSSDELKVLLPEPFMTFSDYLEGMYEADAKIERESVFELLASGDDLFKKIDEIRNNYSGVEFDVEKFQEFCKELNVNPMLIGHVIEAIFSQKAGVTVTGLGTLSPEMGASVALSNTSVDTCEDMDVTEDMEDIVLMADKSHSYMSPEMARWADVKYDNNKGGLVEYKVGTSMTLPATWAEKGKAVLPLSGICVRKPQFSKPLDEEDDLRLSNMNFFKVSDLKLKKTITPVVYTGTIRERQMKNYIDYLSASLGSTLGNLERIVRSDWNGTEESMQTFGLYDCEKCKWLLLPAEKKHAWAVVLASDDTTRIIFLSYDESGSPIIDKRNWKRFAVCSETKVYSVIRSLEVLNKEAIVDPGVHITLVDGVPGCGKTAEIIARVNWKTDLVLTPGREAAAMIRRRACALHKSPVATSDNVRTFDSFVMNKKIFKFDAVYVDEGLMVHTGLLNFALKISGCKKAFVFGDAKQIPFINRVMNFDYPKELRTLIVDNVERRYVTHRCPRDVTSFLNTIYKAAVATTSPVVHSVKAIKVSGAGILRPELTKIKGKIITFTQSDKQSLIKSGYNDVNTVHEIQGETFEETAVVRATPTPIGLIVRDSPHVLVALTRHTKAMVYYTVVFDAVTSIIVDVEKVDQSILTMFATTVPTK.

The 129 kDa protein encoded by the attenuated CGMMV strain ONBM-2differs from the 129 kDa protein encoded by the wild type CGMMV Ontariostrain (SEQ ID NO:63) at least in that:

position 86 of SEQ ID NO:46 is serine (S, Ser);

position 480 of SEQ ID NO:46 is glycine (G, Gly);

position 534 of SEQ ID NO:46 is phenylalanine (F, Phe);

position 1092 of SEQ ID NO:46 is valine (V, Val); and

position 1124 of SEQ ID NO:46 is valine (V, Val).

The 186 kDa protein encoded by the attenuated CGMMV strain ONBM-2 hasthe following sequence.

(SEQ ID NO: 47) MANINEQINNQRDAAASGRNNLVSQLASKRVYDEAVRSLDHQDRRPKMNFSRVVSTEHTRLVTDAYPEFSISFTATKNSVHSLAGSLRLLELEYMMMQVPYGSPCYDIGGNYTQHLFKGRSYVHCCNPCLDLKDVARNVMYNDMVTQHVQRHKGSGGCRPLPTFQIDAFRRYDNSPCAVTCSDVFQECSYDFGSGRDNHAVSLHSIYDIPYSSIGPALHRKNVRVCYAAFHFSEALLLGSPVGNLNSIGAQFRVDGDDVHFLFSEESTLHYTHSLENIKLIVMRTYFPADDRYVYIKEFMVKRVDTFFFRLVRADTHMLHKSVGHYSKSKSEYFALNTPPIFQDKATFSVWFPEAKRKVLIPKFELSRFLSGNVKISRMLVDADFVHTIINHISTYDNKALVWKNVQSFVESIRSRVIVNGVSVKSEWNVPVDQLTDISFSIFLLVKVRKVQIELMSDKVVIEARGLLRRFADSLKSAVGGLGDCVYDALVQTGWFDTSSDELKVLLPEPFMTFSDYLEGMYEADAKIERESVFELLASGDDLFKKIDEIRNNYSGVEFDVEKFQEFCKELNVNPMLIGHVIEAIFSQKAGVTVTGLGTLSPEMGASVALSNTSVDTCEDMDVTEDMEDIVLMADKSHSYMSPEMARWADVKYDNNKGGLVEYKVGTSMTLPATWAEKGKAVLPLSGICVRKPQFSKPLDEEDDLRLSNMNFFKVSDLKLKKTITPVVYTGTIRERQMKNYIDYLSASLGSTLGNLERIVRSDWNGTEESMQTFGLYDCEKCKWLLLPAEKKHAWAVVLASDDTTRIIFLSYDESGSPIIDKRNWKRFAVCSETKVYSVIRSLEVLNKEAIVDPGVHITLVDGVPGCGKTAEIIARVNWKTDLVLTPGREAAAMIRRRACALHKSPVATSDNVRTFDSFVMNKKIFKFDAVYVDEGLMVHTGLLNFALKISGCKKAFVFGDAKQIPFINRVMNFDYPKELRTLIVDNVERRYVTHRCPRDVTSFLNTIYKAAVATTSPVVHSVKAIKVSGAGILRPELTKIKGKIITFTQSDKQSLIKSGYNDVNTVHEIQGETFEETAVVRATPTPIGLIVRDSPHVLVALTRHTKAMVYYTVVFDAVTSIIVDVEKVDQSILTMFATTVPTKXQLMQNSLYVHRDIFLPVSKTGFYTDMQEFYDRCLPGNSFVLNDFDAVTMRLRDNEFNLQPCRLTLSNLDPVPALVKSEAQNFLIPVLRTACERPRIPGLLENLVAMIKRNMNTPDLAGTVDITNMSISIVDNFFSSFVRDEVLLDHLDCVRASSIQSFSDWFSCQPTSAVGQLANFNFIDLPAFDTYMHMIKRQPKSRLDTSIQSEYPALQTIVYHLKVVNAVFGPVFKYLTTKFLSMVDSSKFFFYTRKKSEDLQEFFSDLSSHSDYEILELDVSKYDKSQSDFHFSIEMAIWEKLGLDDILAWMWSMGHKRTILQDFQAGIKTLIYYQRKSGDVTTFIGNTFIIAACVASMLPLDKCFKASFCGDDSLIYLPKGLEYPDIQATANLVWNFEAKLFRKKYGYFCGKYIIHHANGCIVYPDPLKLISKLGNKSLVGYEHVEEFRISLLDVAHSLFNGAYFHLLDDAIHELFPNAGGCSFVINCLCKYLSDKRLFRSLYIDVSK.

The 186 kDa protein encoded by the attenuated CGMMV strain ONBM-2differs from the 186 kDa protein encoded by the wild type CGMMV Ontariostrain (SEQ ID NO:64) at least in that:

position 86 of SEQ ID NO:47 is serine (S, Ser);

position 480 of SEQ ID NO:47 is glycine (G, Gly);

position 534 of SEQ ID NO:47 is phenylalanine (F, Phe);

position 1092 of SEQ ID NO:47 is valine (V, Val);

position 1124 of SEQ ID NO:47 is valine (V, Val);

position 1157 of SEQ ID NO:47 is aspartic acid (D, Asp);

position 1362 of SEQ ID NO:47 is leucine (L, Leu); and

position 1397 of SEQ ID NO:47 is serine (S, Ser).

The coat protein encoded by the attenuated CGMMV ONBM-2 strain has thesequence of SEQ ID NO:32 and differs from the coat protein encoded bythe wild type CGMMV Ontario strain (SEQ ID NO:65) at least in thatposition 156 of SEQ ID NO:32 is valine (V, Val).

Mutant CGMMV Ontario Strain ONBM-3

Directed mutation of the cDNA genome of the cloned CGMMV Ontario strain(Example 1) was carried out as described above to introduce mutationscorresponding to those induced in the cDNA genome of the cloned CGMMVOntario strain mutants ONB and ONM (c.315G>A; c.1498A>G; c.1660C>T;c.3430C>T; c.3528A>G; c.4144C>T; c.4248C>T; and c.6228C>T) in additionto the mutation c.4969G>A introduced by randomly replacing G by A duringPCR amplification. These mutations resulted in amino acid substitutionsin the encoded viral proteins (G86S, E480G, S534F and A1124V in the 129kDa protein; G86S, E480G, S534F, A1124V, N1157D, P1362L, P1397S andR1637H in the 186 kDa protein; and A156V in the coat protein). Theresulting mutant CGMMV strain was designated Ontario strain ONBM-3.

The cDNA genome sequence of CGMMV strain ONBM-3 is shown below.

(SEQ ID NO: 48) GTTTTAATTTTTAAAATTAAACAAACAACAACAACAACAACAAACAATTTAAAACAACAATGGCAAACATTAATGAACAAATCAACAACCAACGCGACGCCGCGGCCAGCGGGAGAAACAATCTCGTTAGCCAATTGGCGTCAAAAAGGGTGTATGACGAGGCTGTTCGCTCGTTGGATCATCAAGACAGACGCCCAAAAATGAACTTTTCTCGTGTGGTCAGCACAGAGCACACCAGGCTTGTAACTGATGCGTATCCGGAGTTTTCGATTAGCTTTACCGCCACCAAGAACTCTGTACACTCCCTTGCGGGTAGTCTGAGGCTCCTTGAACTGGAATATATGATGATGCAAGTGCCCTACGGCTCACCTTGTTATGATATCGGCGGTAACTATACGCAGCACTTGTTCAAAGGTAGATCATATGTGCATTGCTGCAATCCGTGCCTGGATCTTAAAGATGTTGCGAGGAACGTGATGTATAACGATATGGTCACACAACATGTACAGAGGCACAAGGGATCTGGCGGGTGCAGACCTCTTCCAACTTTTCAGATAGATGCATTCAGGAGGTACGATAATTCTCCCTGTGCGGTCACCTGTTCAGACGTTTTCCAAGAGTGTTCCTATGATTTTGGGAGCGGTAGGGATAATCATGCAGTCTCGCTGCATTCAATCTACGATATCCCTTATTCTTCGATCGGACCTGCTCTTCATAGGAAGAACGTGCGAGTTTGTTATGCAGCCTTTCACTTCTCGGAGGCATTGCTTTTAGGTTCACCTGTAGGTAATTTAAATAGTATTGGGGCTCAGTTTAGGGTCGATGGTGATGATGTGCATTTTCTTTTTAGTGAAGAGTCTACTTTGCATTATACTCATAGTTTAGAAAATATCAAATTAATTGTGATGCGTACTTATTTTCCTGCTGATGATAGGTACGTGTATATTAAGGAGTTTATGGTCAAGCGTGTGGATACTTTCTTCTTTAGGTTGGTCAGAGCAGACACACATATGCTTCATAAATCTGTGGGGCACTATTCAAAATCGAAATCTGAGTACTTTGCGCTGAATACCCCTCCGATCTTCCAAGACAAAGCCACGTTTTCTGTGTGGTTTCCTGAGGCGAAGCGTAAGGTGTTGATACCCAAGTTTGAACTTTCAAGATTCCTTTCTGGGAATGTGAAAATCTCTAGGATGCTTGTCGATGCTGATTTCGTCCATACCATTATTAATCACATTAGCACGTATGATAATAAGGCCTTAGTGTGGAAGAATGTTCAGTCCTTTGTGGAATCTATACGCTCAAGAGTAATTGTAAACGGAGTTTCGGTGAAATCTGAATGGAACGTACCGGTTGATCAGCTCACTGATATCTCGTTCTCGATATTCCTTCTCGTGAAGGTTAGGAAGGTACAGATCGAGTTAATGTCTGATAAAGTTGTAATCGAGGCGAGGGGCTTGCTCCGGAGGTTCGCAGACAGTCTTAAATCCGCCGTAGGAGGACTAGGTGATTGCGTCTATGATGCTCTAGTTCAAACCGGCTGGTTTGATACCTCTAGCGACGAACTGAAAGTTTTGCTACCTGAACCGTTTATGACCTTTTCGGATTATCTTGAAGGGATGTACGAGGCAGATGCAAAGATCGAGAGAGAGAGTGTCTTTGAGTTGCTCGCTTCCGGTGACGATTTGTTCAAGAAAATCGATGAGATAAGAAACAATTACAGTGGAGTCGAATTTGATGTAGAGAAATTCCAGGAATTTTGCAAGGAACTGAATGTTAATCCTATGCTAATTGGCCATGTTATCGAAGCTATTTTTTCGCAGAAAGCTGGGGTGACAGTAACGGGTCTGGGTACCCTCTCTCCTGAGATGGGTGCTTCTGTTGCGTTATCCAATACCTCTGTAGATACATGTGAAGATATGGATGTAACTGAAGATATGGAGGATATAGTGTTGATGGCGGACAAGAGTCATTCTTACATGTCCCCAGAAATGGCGAGATGGGCTGATGTAAAATACGACAACAATAAAGGGGGCCTGGTCGAATACAAAGTCGGAACCTCGATGACTTTACCTGCCACCTGGGCAGAGAAGGGTAAGGCTGTCTTACCGTTGTCGGGGATCTGTGTGAGGAAACCCCAATTTTCGAAGCCGCTTGATGAGGAAGACGACTTGAGGTTATCAAACATGAATTTCTTTAAGGTGAGCGATCTGAAGTTGAAGAAAACTATCACTCCAGTTGTTTACACTGGGACCATTCGAGAGAGGCAAATGAAGAATTATATTGATTACTTATCGGCCTCTCTTGGTTCTACGCTGGGTAATCTGGAGAGAATTGTGCGGAGTGATTGGAACGGTACCGAGGAGAGTATGCAAACGTTCGGGTTGTATGACTGCGAAAAGTGCAAGTGGTTACTGTTACCAGCCGAAAAGAAGCACGCATGGGCTGTGGTTCTGGCAAGTGATGATACCACTCGCATAATCTTCCTCTCATATGACGAATCTGGTTCTCCCATAATTGATAAGAGAAACTGGAAGCGATTTGCTGTTTGCTCTGAGACCAAAGTCTATAGCGTAATTCGTAGTTTAGAGGTACTAAATAAGGAAGCAATAGTCGACCCCGGGGTTCATATAACATTAGTTGACGGAGTGCCGGGTTGTGGAAAGACCGCCGAAATTATAGCGAGGGTCAATTGGAAAACCGATCTAGTATTGACTCCCGGGAGGGAGGCGGCTGCTATGATTAGGCGGAGGGCCTGCGCCCTGCACAAGTCACCTGTGGCAACCAGTGACAACGTTAGAACTTTCGATTCTTTTGTGATGAATAAGAAAATCTTCAAGTTTGACGCTGTCTATGTTGACGAGGGTCTGATGGTCCATACGGGTTTACTTAATTTTGCGTTGAAGATCTCAGGTTGTAAAAAGGCCTTCGTCTTTGGTGATGCTAAGCAAATCCCGTTTATAAACAGAGTCATGAATTTTGATTATCCTAAGGAGTTAAGAACTTTAATAGTCGATAATGTAGAGCGTAGGTATGTTACCCATAGGTGTCCTAGAGATGTCACTAGTTTTCTTAATACTATTTACAAAGCCGCTGTCGCTACTACTAGTCCGGTTGTACATTCTGTGAAGGCGATTAAAGTGTCAGGGGCCGGTATTCTGAGGCCCGAGTTGACGAAGATCAAAGGAAAGATAATAACGTTTACTCAATCTGATAAGCAGTCCTTGATCAAGAGTGGGTACAATGACGTGAACACTGTGCATGAAATTCAGGGAGAAACCTTTGAAGAGACGGCGGTTGTGCGTGCCACCCCGACTCCGATAGGTTTAATTGCCCGTGATTCACCACATGTACTAGTGGCCTTAACGAGGCACACTAAGGCAATGGTGTATTATACTGTTGTGTTCGATGCAGTTACAAGTATAATAGTGGATGTGGAAAAGGTCGACCAGTCGATCTTGACTATGTTTGCTACCACTGTGCCTACCAAATAGCAATTAATGCAGAACTCACTGTATGTCCATCGTGATATTTTCCTCCCTGTTAGTAAAACGGGGTTTTATACAGACATGCAGGAGTTCTATGATAGATGCCTTCCTGGGAATTCCTTCGTGCTGAATGATTTCGATGCCGTAACCATGCGGTTGAGGGACAACGAATTTAACCTACAACCTTGTAGGCTAACCTTAAGTAATTTAGATCCAGTACCCGCTTTGGTTAAGAGTGAAGCGCAGAATTTTCTGATTCCCGTTTTGCGTACGGCCTGTGAAAGGCCGCGCATTCCAGGTCTCCTTGAAAATCTTGTAGCTATGATAAAGAGGAATATGAATACTCCTGATCTAGCTGGGACTGTGGATATAACTAATATGTCGATTTCTATAGTAGATAACTTCTTTTCTTCTTTTGTTAGAGACGAGGTTTTGCTTGATCATTTAGATTGTGTTAGGGCTAGTTCCATTCAAAGTTTTTCTGATTGGTTTTCGTGTCAGCCAACCTCGGCGGTTGGTCAATTAGCTAATTTCAATTTCATAGATTTGCCTGCCTTTGATACTTATATGCACATGATTAAGCGGCAGCCCAAGAGTCGGTTGGATACTTCGATTCAGTCTGAATATCCGGCCTTGCAAACTATTGTTTATCACCTTAAAGTGGTAAATGCAGTTTTCGGTCCGGTTTTTAAGTATTTGACCACCAAGTTTCTTAGCATGGTAGATAGTTCTAAGTTTTTCTTTTACACTAGGAAAAAATCAGAAGATCTGCAGGAATTTTTCTCAGATCTCTCTTCCCATTCTGATTATGAGATTCTTGAGCTGGATGTTTCTAAATATGACAAGTCACAATCCGATTTCCATTTCTCTATTGAGATGGCAATTTGGGAAAAATTGGGGCTGGACGATATTTTGGCTTGGATGTGGTCTATGGGTCACAAGAGAACTATACTGCAAGATTTCCAAGCCGGGATAAAGACGCTCATTTACTATCAACGGAAGTCTGGTGATGTAACTACTTTCATAGGTAATACCTTTATTATCGCAGCGTGTGTAGCTAGTATGTTGCCGTTAGACAAGTGTTTTAAAGCTAGTTTTTGTGGTGATGATTCGCTGATCTACCTTCCTAAGGGTTTGGAGTATCCTGATATACAGGCTACTGCCAACTTGGTTTGGAATTTTGAGGCGAAACTTTTCCGAAAGAAGTATGGTTACTTCTGTGGGAAGTATATAATTCACCATGCCAACGGCTGTATTGTTTACCCTGACCCTTTAAAATTAATTAGTAAATTAGGTAATAAGAGTCTTGTAGGGTATGAGCATGTTGAGGAGTTTCGTATATCTCTCCTCGACGTCGCTCATAGTTTGTTTAATGGTGCTTATTTCCATTTACTCGACGATGCAATCCACGAATTATTTCCTAACGCTGGGGGTTGCAGTTTTGTAATTAATTGTTTGTGCAAGTATTTGAGTGATAAGCACCTTTTCCGTAGTCTTTATATAGATGTCTCTAAGTAAGGTGTCGGTCGAGAACTCATTGAAACCCGAGAAGTTTGTTAAAATCTCTTGGGTCGATAAGTTGCTCCCTAACTATTTTTCCATTCTTAAGTATTTATCTATAACTGACTTTAGCGTAGTTAAAGCTCAGAGCTATGAATCCCTCGTGCCTGTCAAGTTGTTGCGTGGTGTTGATCTTACAAAACACCTTTATGTCACATTGTTGGGCGTTGTGGTTTCTGGTGTATGGAACGTACCGGAATCCTGTAGGGGTGGTGCTACTGTTGCTCTGGTTGACACAAGGATGCATTCTGTTGCAGAGGGAACTATATGCAAATTTTCAGCTCCCGCCACCGTCCGCGAATTCTCTGTTAGGTTCATACCTAACTATTCTGTCGTGGCTGCGGATGCCCTTCGCGATCCTTGGTCTTTATTTGTGAGACTCTCTAATGTAGGGATTAAAGATGGTTTCCATCCTTTGACCTTAGAGGTCGCTTGTTTAGTCGCTACAACTAACTCTATTATCAAAAAGGGTCTTAGAGCTTCTGTAGTCGAGTCTGTCGTCTCTTCCGATCAGTCCATTGTCCTAGATTCTTTATCCGAGAAAGTTGAACCTTTCTTTGATAAAGTTCCTATTTCGGCGGCTGTGATGGCAAGAGACCCCAGTTATAGGTCTAGGTCGCAGTCTGTCGGTGGTCGTGGTAAGCGGCATTCTAAACCTCCAAATCGGAGGTTGGACTCTGCTTCTGAAGAGTCCAGTTCTGTTTCTTTCGAAGATGGCTTACAATCCGATCACACCTAGCAAACTTATTGCGTTTAGTGCTTCTTATGTTCCCGTCAGGACTTTACTTAATTTTCTAGTTGCTTCACAAGGTACCGCCTTCCAGACTCAAGCGGGAAGAGATTCTTTCCGCGAGTCCCTGTCTGCGTTACCCTCGTCTGTCGTAGATATTAATTCTAGGTTCCCAAATGCGGGTTTTTACGCTTTCCTCAACGGTCCTGTGTTGAGGCCTATCTTCGTTTCGCTTCTTAGCTCTACGGATACGCGTAATAGGGTCATTGAGGTTGTAGATCCTAGCAATCCTACGACTGCTGAGTCGCTTAACGCTGTAAAGCGTACTGATGACGCATCTACGGCCGCTAGGGCTGAAATAGATAATTTAATAGAGTCTATTTCTAAGGGTTTTGATGTTTATGATAGGGCTTCATTTGAAGCCGCGTTTTCGGTAGTCTGGTCAGAGGTTACCACCTCGAAAGCTTAGCTTCGAGGGTCTTCTGATGGTGGTGCACACCAAAGTGCATAGTGCTTTCCCGTTCACTTAAATCGAACGGTTTGCTCATTGGTTTGCGGAAACCTCTCACGTGTGGCGTTGAAGTTTCTATGGGCAGTAATTCTGCAAGGGGTTCGAATCCCCCCTTTCCCCGGGTAGGGGCCCA.

The cDNA genome sequence of the attenuated CGMMV strain ONBM-3 differsfrom the cDNA genome sequence of the wild type CGMMV Ontario strain (SEQID NO:18) at least in that:

the nucleotide at position 315 of SEQ ID NO:48 is A;

the nucleotide at position 1498 of SEQ ID NO:48 is G;

the nucleotide at position 1660 of SEQ ID NO:48 is T;

the nucleotide at position 3430 of SEQ ID NO:48 is T;

the nucleotide at position 3528 of SEQ ID NO:48 is G;

the nucleotide at position 4144 of SEQ ID NO:48 is T;

the nucleotide at position 4248 of SEQ ID NO:48 is T;

the nucleotide at position 4969 of SEQ ID NO:48 is A; and

the nucleotide at position 6228 of SEQ ID NO:48 is T.

The 129 kDa protein encoded by the attenuated CGMMV strain ONBM-3 hasthe following sequence.

(SEQ ID NO: 49) MANINEQINNQRDAAASGRNNLVSQLASKRVYDEAVRSLDHQDRRPKMNFSRVVSTEHTRLVTDAYPEFSISFTATKNSVHSLAGSLRLLELEYMMMQVPYGSPCYDIGGNYTQHLFKGRSYVHCCNPCLDLKDVARNVMYNDMVTQHVQRHKGSGGCRPLPTFQIDAFRRYDNSPCAVTCSDVFQECSYDFGSGRDNHAVSLHSIYDIPYSSIGPALHRKNVRVCYAAFHFSEALLLGSPVGNLNSIGAQFRVDGDDVHFLFSEESTLHYTHSLENIKLIVMRTYFPADDRYVYIKEFMVKRVDTFFFRLVRADTHMLHKSVGHYSKSKSEYFALNTPPIFQDKATFSVWFPEAKRKVLIPKFELSRFLSGNVKISRMLVDADFVHTIINHISTYDNKALVWKNVQSFVESIRSRVIVNGVSVKSEWNVPVDQLTDISFSIFLLVKVRKVQIELMSDKVVIEARGLLRRFADSLKSAVGGLGDCVYDALVQTGWFDTSSDELKVLLPEPFMTFSDYLEGMYEADAKIERESVFELLASGDDLFKKIDEIRNNYSGVEFDVEKFQEFCKELNVNPMLIGHVIEAIFSQKAGVTVTGLGTLSPEMGASVALSNTSVDTCEDMDVTEDMEDIVLMADKSHSYMSPEMARWADVKYDNNKGGLVEYKVGTSMTLPATWAEKGKAVLPLSGICVRKPQFSKPLDEEDDLRLSNMNFFKVSDLKLKKTITPVVYTGTIRERQMKNYIDYLSASLGSTLGNLERIVRSDWNGTEESMQTFGLYDCEKCKWLLLPAEKKHAWAVVLASDDTTRIIFLSYDESGSPIIDKRNWKRFAVCSETKVYSVIRSLEVLNKEAIVDPGVHITLVDGVPGCGKTAEIIARVNWKTDLVLTPGREAAAMIRRRACALHKSPVATSDNVRTFDSFVMNKKIFKFDAVYVDEGLMVHTGLLNFALKISGCKKAFVFGDAKQIPFINRVMNFDYPKELRTLIVDNVERRYVTHRCPRDVTSFLNTIYKAAVATTSPVVHSVKAIKVSGAGILRPELTKIKGKIITFTQSDKQSLIKSGYNDVNTVHEIQGETFEETAVVRATPTPIGLIARDSPHVLVALTRHTKAMVYYTVVFDAVTSIIVDVEKVDQSILTMFATTVPTK.

The 129 kDa protein encoded by the attenuated CGMMV strain ONBM-3differs from the 129 kDa protein encoded by the wild type CGMMV Ontariostrain (SEQ ID NO:63) at least in that:

position 86 of SEQ ID NO:49 is serine (S, Ser);

position 480 of SEQ ID NO:49 is glycine (G, Gly);

position 534 of SEQ ID NO:49 is phenylalanine (F, Phe); and

position 1124 of SEQ ID NO:49 is valine (V, Val).

The 186 kDa protein encoded by the attenuated CGMMV strain ONBM-3 hasthe following sequence.

(SEQ ID NO: 50) MANINEQINNQRDAAASGRNNLVSQLASKRVYDEAVRSLDHQDRRPKMNFSRVVSTEHTRLVTDAYPEFSISFTATKNSVHSLAGSLRLLELEYMMMQVPYGSPCYDIGGNYTQHLFKGRSYVHCCNPCLDLKDVARNVMYNDMVTQHVQRHKGSGGCRPLPTFQIDAFRRYDNSPCAVTCSDVFQECSYDFGSGRDNHAVSLHSIYDIPYSSIGPALHRKNVRVCYAAFHFSEALLLGSPVGNLNSIGAQFRVDGDDVHFLFSEESTLHYTHSLENIKLIVMRTYFPADDRYVYIKEFMVKRVDTFFFRLVRADTHMLHKSVGHYSKSKSEYFALNTPPIFQDKATFSVWFPEAKRKVLIPKFELSRFLSGNVKISRMLVDADFVHTIINHISTYDNKALVWKNVQSFVESIRSRVIVNGVSVKSEWNVPVDQLTDISFSIFLLVKVRKVQIELMSDKVVIEARGLLRRFADSLKSAVGGLGDCVYDALVQTGWFDTSSDELKVLLPEPFMTFSDYLEGMYEADAKIERESVFELLASGDDLFKKIDEIRNNYSGVEFDVEKFQEFCKELNVNPMLIGHVIEAIFSQKAGVTVTGLGTLSPEMGASVALSNTSVDTCEDMDVTEDMEDIVLMADKSHSYMSPEMARWADVKYDNNKGGLVEYKVGTSMTLPATWAEKGKAVLPLSGICVRKPQFSKPLDEEDDLRLSNMNFFKVSDLKLKKTITPVVYTGTIRERQMKNYIDYLSASLGSTLGNLERIVRSDWNGTEESMQTFGLYDCEKCKWLLLPAEKKHAWAVVLASDDTTRIIFLSYDESGSPIIDKRNWKRFAVCSETKVYSVIRSLEVLNKEAIVDPGVHITLVDGVPGCGKTAEIIARVNWKTDLVLTPGREAAAMIRRRACALHKSPVATSDNVRTFDSFVMNKKIFKFDAVYVDEGLMVHTGLLNFALKISGCKKAFVFGDAKQIPFINRVMNFDYPKELRTLIVDNVERRYVTHRCPRDVTSFLNTIYKAAVATTSPVVHSVKAIKVSGAGILRPELTKIKGKIITFTQSDKQSLIKSGYNDVNTVHEIQGETFEETAVVRATPTPIGLIARDSPHVLVALTRHTKAMVYYTVVFDAVTSIIVDVEKVDQSILTMFATTVPTKXQLMQNSLYVHRDIFLPVSKTGFYTDMQEFYDRCLPGNSFVLNDFDAVTMRLRDNEFNLQPCRLTLSNLDPVPALVKSEAQNFLIPVLRTACERPRIPGLLENLVAMIKRNMNTPDLAGTVDITNMSISIVDNFFSSFVRDEVLLDHLDCVRASSIQSFSDWFSCQPTSAVGQLANFNFIDLPAFDTYMHMIKRQPKSRLDTSIQSEYPALQTIVYHLKVVNAVFGPVFKYLTTKFLSMVDSSKFFFYTRKKSEDLQEFFSDLSSHSDYEILELDVSKYDKSQSDFHFSIEMAIWEKLGLDDILAWMWSMGHKRTILQDFQAGIKTLIYYQRKSGDVTTFIGNTFIIAACVASMLPLDKCFKASFCGDDSLIYLPKGLEYPDIQATANLVWNFEAKLFRKKYGYFCGKYIIHHANGCIVYPDPLKLISKLGNKSLVGYEHVEEFRISLLDVAHSLFNGAYFHLLDDAIHELFPNAGGCSFVINCLCKYLSDKHLFRSLYIDVSK.

The 186 kDa protein encoded by the attenuated CGMMV strain ONBM-3differs from the 186 kDa protein encoded by the wild type CGMMV Ontariostrain (SEQ ID NO:64) at least in that:

position 86 of SEQ ID NO:50 is serine (5, Ser);

position 480 of SEQ ID NO:50 is glycine (G, Gly);

position 534 of SEQ ID NO:50 is phenylalanine (F, Phe);

position 1124 of SEQ ID NO:50 is valine (V, Val);

position 1157 of SEQ ID NO:50 is aspartic acid (D, Asp);

position 1362 of SEQ ID NO:50 is leucine (L, Leu);

position 1397 of SEQ ID NO:50 is serine (S, Ser); and

position 1637 of SEQ ID NO:50 is histidine (H, His).

The coat protein encoded by the attenuated CGMMV strain ONBM-3 has thesequence of SEQ ID NO:32 and differs from the coat protein encoded bythe wild type CGMMV Ontario strain (SEQ ID NO:65) at least in thatposition 156 of SEQ ID NO:32 is valine (V, Val).

Mutant CGMMV Ontario Strain ONAL-1

Directed mutation of the cDNA genome of the cloned CGMMV Ontario strain(Example 1) was carried out as described above, using the mutagenicprimers listed in Table 5 to introduce the mutation c.3334C>T.Nucleotide residues indicated in bold indicate sites of mutation. Thismutation resulted in an A1092V amino acid substitution in the encodedviral 129 kDa and 186 kDa proteins. The resulting mutant CGMMV strainwas designated Ontario strain ONAL-1.

TABLE 5 Primers used to produce mutant CGMMV Ontario strain ONAL-1Sequence Primer Sequence (5′ to 3′) IdentifierTGTGGTGAATCACGGACAATTAAACCTATCGGAGTCG SEQ ID NO: 51CGACTCCGATAGGTTTAATTGTCCGTGATTCACCACA SEQ ID NO: 52

The cDNA genome sequence of CGMMV strain ONAL-1 is shown below.

(SEQ ID NO: 53) GTTTTAATTTTTAAAATTAAACAAACAACAACAACAACAACAAACAATTTAAAACAACAATGGCAAACATTAATGAACAAATCAACAACCAACGCGACGCCGCGGCCAGCGGGAGAAACAATCTCGTTAGCCAATTGGCGTCAAAAAGGGTGTATGACGAGGCTGTTCGCTCGTTGGATCATCAAGACAGACGCCCAAAAATGAACTTTTCTCGTGTGGTCAGCACAGAGCACACCAGGCTTGTAACTGATGCGTATCCGGAGTTTTCGATTAGCTTTACCGCCACCAAGAACTCTGTACACTCCCTTGCGGGTGGTCTGAGGCTCCTTGAACTGGAATATATGATGATGCAAGTGCCCTACGGCTCACCTTGTTATGATATCGGCGGTAACTATACGCAGCACTTGTTCAAAGGTAGATCATATGTGCATTGCTGCAATCCGTGCCTGGATCTTAAAGATGTTGCGAGGAACGTGATGTATAACGATATGGTCACACAACATGTACAGAGGCACAAGGGATCTGGCGGGTGCAGACCTCTTCCAACTTTTCAGATAGATGCATTCAGGAGGTACGATAATTCTCCCTGTGCGGTCACCTGTTCAGACGTTTTCCAAGAGTGTTCCTATGATTTTGGGAGCGGTAGGGATAATCATGCAGTCTCGCTGCATTCAATCTACGATATCCCTTATTCTTCGATCGGACCTGCTCTTCATAGGAAGAACGTGCGAGTTTGTTATGCAGCCTTTCACTTCTCGGAGGCATTGCTTTTAGGTTCACCTGTAGGTAATTTAAATAGTATTGGGGCTCAGTTTAGGGTCGATGGTGATGATGTGCATTTTCTTTTTAGTGAAGAGTCTACTTTGCATTATACTCATAGTTTAGAAAATATCAAATTAATTGTGATGCGTACTTATTTTCCTGCTGATGATAGGTACGTGTATATTAAGGAGTTTATGGTCAAGCGTGTGGATACTTTCTTCTTTAGGTTGGTCAGAGCAGACACACATATGCTTCATAAATCTGTGGGGCACTATTCAAAATCGAAATCTGAGTACTTTGCGCTGAATACCCCTCCGATCTTCCAAGACAAAGCCACGTTTTCTGTGTGGTTTCCTGAGGCGAAGCGTAAGGTGTTGATACCCAAGTTTGAACTTTCAAGATTCCTTTCTGGGAATGTGAAAATCTCTAGGATGCTTGTCGATGCTGATTTCGTCCATACCATTATTAATCACATTAGCACGTATGATAATAAGGCCTTAGTGTGGAAGAATGTTCAGTCCTTTGTGGAATCTATACGCTCAAGAGTAATTGTAAACGGAGTTTCGGTGAAATCTGAATGGAACGTACCGGTTGATCAGCTCACTGATATCTCGTTCTCGATATTCCTTCTCGTGAAGGTTAGGAAGGTACAGATCGAGTTAATGTCTGATAAAGTTGTAATCGAGGCGAGGGGCTTGCTCCGGAGGTTCGCAGACAGTCTTAAATCCGCCGTAGAAGGACTAGGTGATTGCGTCTATGATGCTCTAGTTCAAACCGGCTGGTTTGATACCTCTAGCGACGAACTGAAAGTTTTGCTACCTGAACCGTTTATGACCTTTTCGGATTATCTTGAAGGGATGTACGAGGCAGATGCAAAGATCGAGAGAGAGAGTGTCTCTGAGTTGCTCGCTTCCGGTGACGATTTGTTCAAGAAAATCGATGAGATAAGAAACAATTACAGTGGAGTCGAATTTGATGTAGAGAAATTCCAGGAATTTTGCAAGGAACTGAATGTTAATCCTATGCTAATTGGCCATGTTATCGAAGCTATTTTTTCGCAGAAAGCTGGGGTGACAGTAACGGGTCTGGGTACCCTCTCTCCTGAGATGGGTGCTTCTGTTGCGTTATCCAATACCTCTGTAGATACATGTGAAGATATGGATGTAACTGAAGATATGGAGGATATAGTGTTGATGGCGGACAAGAGTCATTCTTACATGTCCCCAGAAATGGCGAGATGGGCTGATGTAAAATACGACAACAATAAAGGGGGCCTGGTCGAATACAAAGTCGGAACCTCGATGACTTTACCTGCCACCTGGGCAGAGAAGGGTAAGGCTGTCTTACCGTTGTCGGGGATCTGTGTGAGGAAACCCCAATTTTCGAAGCCGCTTGATGAGGAAGACGACTTGAGGTTATCAAACATGAATTTCTTTAAGGTGAGCGATCTGAAGTTGAAGAAAACTATCACTCCAGTTGTTTACACTGGGACCATTCGAGAGAGGCAAATGAAGAATTATATTGATTACTTATCGGCCTCTCTTGGTTCTACGCTGGGTAATCTGGAGAGAATTGTGCGGAGTGATTGGAACGGTACCGAGGAGAGTATGCAAACGTTCGGGTTGTATGACTGCGAAAAGTGCAAGTGGTTACTGTTACCAGCCGAAAAGAAGCACGCATGGGCTGTGGTTCTGGCAAGTGATGATACCACTCGCATAATCTTCCTCTCATATGACGAATCTGGTTCTCCCATAATTGATAAGAGAAACTGGAAGCGATTTGCTGTTTGCTCTGAGACCAAAGTCTATAGCGTAATTCGTAGTTTAGAGGTACTAAATAAGGAAGCAATAGTCGACCCCGGGGTTCATATAACATTAGTTGACGGAGTGCCGGGTTGTGGAAAGACCGCCGAAATTATAGCGAGGGTCAATTGGAAAACCGATCTAGTATTGACTCCCGGGAGGGAGGCGGCTGCTATGATTAGGCGGAGGGCCTGCGCCCTGCACAAGTCACCTGTGGCAACCAGTGACAACGTTAGAACTTTCGATTCTTTTGTGATGAATAAGAAAATCTTCAAGTTTGACGCTGTCTATGTTGACGAGGGTCTGATGGTCCATACGGGTTTACTTAATTTTGCGTTGAAGATCTCAGGTTGTAAAAAGGCCTTCGTCTTTGGTGATGCTAAGCAAATCCCGTTTATAAACAGAGTCATGAATTTTGATTATCCTAAGGAGTTAAGAACTTTAATAGTCGATAATGTAGAGCGTAGGTATGTTACCCATAGGTGTCCTAGAGATGTCACTAGTTTTCTTAATACTATTTACAAAGCCGCTGTCGCTACTACTAGTCCGGTTGTACATTCTGTGAAGGCGATTAAAGTGTCAGGGGCCGGTATTCTGAGGCCCGAGTTGACGAAGATCAAAGGAAAGATAATAACGTTTACTCAATCTGATAAGCAGTCCTTGATCAAGAGTGGGTACAATGACGTGAACACTGTGCATGAAATTCAGGGAGAAACCTTTGAAGAGACGGCGGTTGTGCGTGCCACCCCGACTCCGATAGGTTTAATTGTCCGTGATTCACCACATGTACTAGTGGCCTTAACGAGGCACACTAAGGCAATGGTGTATTATACTGTTGTGTTCGATGCAGTTACAAGTATAATAGCGGATGTGGAAAAGGTCGACCAGTCGATCTTGACTATGTTTGCTACCACTGTGCCTACCAAATAGCAATTAATGCAGAACTCACTGTATGTCCATCGTAATATTTTCCTCCCTGTTAGTAAAACGGGGTTTTATACAGACATGCAGGAGTTCTATGATAGATGCCTTCCTGGGAATTCCTTCGTGCTGAATGATTTCGATGCCGTAACCATGCGGTTGAGGGACAACGAATTTAACCTACAACCTTGTAGGCTAACCTTAAGTAATTTAGATCCAGTACCCGCTTTGGTTAAGAGTGAAGCGCAGAATTTTCTGATTCCCGTTTTGCGTACGGCCTGTGAAAGGCCGCGCATTCCAGGTCTCCTTGAAAATCTTGTAGCTATGATAAAGAGGAATATGAATACTCCTGATCTAGCTGGGACTGTGGATATAACTAATATGTCGATTTCTATAGTAGATAACTTCTTTTCTTCTTTTGTTAGAGACGAGGTTTTGCTTGATCATTTAGATTGTGTTAGGGCTAGTTCCATTCAAAGTTTTTCTGATTGGTTTTCGTGTCAGCCAACCTCGGCGGTTGGTCAATTAGCTAATTTCAATTTCATAGATTTGCCTGCCTTTGATACTTATATGCACATGATTAAGCGGCAGCCCAAGAGTCGGTTGGATACTTCGATTCAGTCTGAATATCCGGCCTTGCAAACTATTGTTTATCACCCTAAAGTGGTAAATGCAGTTTTCGGTCCGGTTTTTAAGTATTTGACCACCAAGTTTCTTAGCATGGTAGATAGTTCTAAGTTTTTCTTTTACACTAGGAAAAAACCAGAAGATCTGCAGGAATTTTTCTCAGATCTCTCTTCCCATTCTGATTATGAGATTCTTGAGCTGGATGTTTCTAAATATGACAAGTCACAATCCGATTTCCATTTCTCTATTGAGATGGCAATTTGGGAAAAATTGGGGCTGGACGATATTTTGGCTTGGATGTGGTCTATGGGTCACAAGAGAACTATACTGCAAGATTTCCAAGCCGGGATAAAGACGCTCATTTACTATCAACGGAAGTCTGGTGATGTAACTACTTTCATAGGTAATACCTTTATTATCGCAGCGTGTGTAGCTAGTATGTTGCCGTTAGACAAGTGTTTTAAAGCTAGTTTTTGTGGTGATGATTCGCTGATCTACCTTCCTAAGGGTTTGGAGTATCCTGATATACAGGCTACTGCCAACTTGGTTTGGAATTTTGAGGCGAAACTTTTCCGAAAGAAGTATGGTTACTTCTGTGGGAAGTATATAATTCACCATGCCAACGGCTGTATTGTTTACCCTGACCCTTTAAAATTAATTAGTAAATTAGGTAATAAGAGTCTTGTAGGGTATGAGCATGTTGAGGAGTTTCGTATATCTCTCCTCGACGTCGCTCATAGTTTGTTTAATGGTGCTTATTTCCATTTACTCGACGATGCAATCCACGAATTATTTCCTAACGCTGGGGGTTGCAGTTTTGTAATTAATTGTTTGTGCAAGTATTTGAGTGATAAGCGCCTTTTCCGTAGTCTTTATATAGATGTCTCTAAGTAAGGTGTCGGTCGAGAACTCATTGAAACCCGAGAAGTTTGTTAAAATCTCTTGGGTCGATAAGTTGCTCCCTAACTATTTTTCCATTCTTAAGTATTTATCTATAACTGACTTTAGCGTAGTTAAAGCTCAGAGCTATGAATCCCTCGTGCCTGTCAAGTTGTTGCGTGGTGTTGATCTTACAAAACACCTTTATGTCACATTGTTGGGCGTTGTGGTTTCTGGTGTATGGAACGTACCGGAATCCTGTAGGGGTGGTGCTACTGTTGCTCTGGTTGACACAAGGATGCATTCTGTTGCAGAGGGAACTATATGCAAATTTTCAGCTCCCGCCACCGTCCGCGAATTCTCTGTTAGGTTCATACCTAACTATTCTGTCGTGGCTGCGGATGCCCTTCGCGATCCTTGGTCTTTATTTGTGAGACTCTCTAATGTAGGGATTAAAGATGGTTTCCATCCTTTGACCTTAGAGGTCGCTTGTTTAGTCGCTACAACTAACTCTATTATCAAAAAGGGTCTTAGAGCTTCTGTAGTCGAGTCTGTCGTCTCTTCCGATCAGTCCATTGTCCTAGATTCTTTATCCGAGAAAGTTGAACCTTTCTTTGATAAAGTTCCTATTTCGGCGGCTGTGATGGCAAGAGACCCCAGTTATAGGTCTAGGTCGCAGTCTGTCGGTGGTCGTGGTAAGCGGCATTCTAAACCTCCAAATCGGAGGTTGGACTCTGCTTCTGAAGAGTCCAGTTCTGTTTCTTTCGAAGATGGCTTACAATCCGATCACACCTAGCAAACTTATTGCGTTTAGTGCTTCTTATGTTCCCGTCAGGACTTTACTTAATTTTCTAGTTGCTTCACAAGGTACCGCCTTCCAGACTCAAGCGGGAAGAGATTCTTTCCGCGAGTCCCTGTCTGCGTTACCCTCGTCTGTCGTAGATATTAATTCTAGGTTCCCAAATGCGGGTTTTTACGCTTTCCTCAACGGTCCTGTGTTGAGGCCTATCTTCGTTTCGCTTCTTAGCTCTACGGATACGCGTAATAGGGTCATTGAGGTTGTAGATCCTAGCAATCCTACGACTGCTGAGTCGCTTAACGCTGTAAAGCGTACTGATGACGCATCTACGGCCGCTAGGGCTGAAATAGATAATTTAATAGAGTCTATTTCTAAGGGTTTTGATGTTTATGATAGGGCTTCATTTGAAGCCGCGTTTTCGGTAGTCTGGTCAGAGGCTACCACCTCGAAAGCTTAGCTTCGAGGGTCTTCTGATGGTGGTGCACACCAAAGTGCATAGTGCTTTCCCGTTCACTTAAATCGAACGGTTTGCTCATTGGTTTGCGGAAACCTCTCACGTGTGGCGTTGAAGTTTCTATGGGCAGTAATTCTGCAAGGGGTTCGAATCCCCCCTTTCCCCGGGTAGGGGCCCA.

The cDNA genome sequence of the attenuated CGMMV strain ONAL-1 differsfrom the cDNA genome sequence of the wild type CGMMV Ontario strain (SEQID NO: 18) at least in that the nucleotide at position 3334 of SEQ IDNO:53 is T.

The 129 kDa protein encoded by the attenuated CGMMV strain ONAL-1 hasthe following sequence.

(SEQ ID NO: 54) MANINEQINNQRDAAASGRNNLVSQLASKRVYDEAVRSLDHQDRRPKMNFSRVVSTEHTRLVTDAYPEFSISFTATKNSVHSLAGGLRLLELEYMMMQVPYGSPCYDIGGNYTQHLFKGRSYVHCCNPCLDLKDVARNVMYNDMVTQHVQRHKGSGGCRPLPTFQIDAFRRYDNSPCAVTCSDVFQECSYDFGSGRDNHAVSLHSIYDIPYSSIGPALHRKNVRVCYAAFHFSEALLLGSPVGNLNSIGAQFRVDGDDVHFLFSEESTLHYTHSLENIKLIVMRTYFPADDRYVYIKEFMVKRVDTFFFRLVRADTHMLHKSVGHYSKSKSEYFALNTPPIFQDKATFSVWFPEAKRKVLIPKFELSRFLSGNVKISRMLVDADFVHTIINHISTYDNKALVWKNVQSFVESIRSRVIVNGVSVKSEWNVPVDQLTDISFSIFLLVKVRKVQIELMSDKVVIEARGLLRRFADSLKSAVEGLGDCVYDALVQTGWFDTSSDELKVLLPEPFMTFSDYLEGMYEADAKIERESVSELLASGDDLFKKIDEIRNNYSGVEFDVEKFQEFCKELNVNPMLIGHVIEAIFSQKAGVTVTGLGTLSPEMGASVALSNTSVDTCEDMDVTEDMEDIVLMADKSHSYMSPEMARWADVKYDNNKGGLVEYKVGTSMTLPATWAEKGKAVLPLSGICVRKPQFSKPLDEEDDLRLSNMNFFKVSDLKLKKTITPVVYTGTIRERQMKNYIDYLSASLGSTLGNLERIVRSDWNGTEESMQTFGLYDCEKCKWLLLPAEKKHAWAVVLASDDTTRIIFLSYDESGSPIIDKRNWKRFAVCSETKVYSVIRSLEVLNKEAIVDPGVHITLVDGVPGCGKTAEIIARVNWKTDLVLTPGREAAAMIRRRACALHKSPVATSDNVRTFDSFVMNKKIFKFDAVYVDEGLMVHTGLLNFALKISGCKKAFVFGDAKQIPFINRVMNFDYPKELRTLIVDNVERRYVTHRCPRDVTSFLNTIYKAAVATTSPVVHSVKAIKVSGAGILRPELTKIKGKIITFTQSDKQSLIKSGYNDVNTVHEIQGETFEETAVVRATPTPIGLIVRDSPHVLVALTRHTKAMVYYTVVFDAVTSIIADVEKVDQSILTMFATTVPTK.

The 129 kDa protein encoded by the attenuated CGMMV strain ONAL-1differs from the 129 kDa protein encoded by the wild type CGMMV Ontariostrain (SEQ ID NO:63) at least in that position 1092 of SEQ ID NO:54 isvaline (V, Val).

The 186 kDa protein encoded by the attenuated CGMMV strain ONAL-1 hasthe following sequence.

(SEQ ID NO: 55) MANINEQINNQRDAAASGRNNLVSQLASKRVYDEAVRSLDHQDRRPKMNFSRVVSTEHTRLVTDAYPEFSISFTATKNSVHSLAGGLRLLELEYMMMQVPYGSPCYDIGGNYTQHLFKGRSYVHCCNPCLDLKDVARNVMYNDMVTQHVQRHKGSGGCRPLPTFQIDAFRRYDNSPCAVTCSDVFQECSYDFGSGRDNHAVSLHSIYDIPYSSIGPALHRKNVRVCYAAFHFSEALLLGSPVGNLNSIGAQFRVDGDDVHFLFSEESTLHYTHSLENIKLIVMRTYFPADDRYVYIKEFMVKRVDTFFFRLVRADTHMLHKSVGHYSKSKSEYFALNTPPIFQDKATFSVWFPEAKRKVLIPKFELSRFLSGNVKISRMLVDADFVHTIINHISTYDNKALVWKNVQSFVESIRSRVIVNGVSVKSEWNVPVDQLTDISFSIFLLVKVRKVQIELMSDKVVIEARGLLRRFADSLKSAVEGLGDCVYDALVQTGWFDTSSDELKVLLPEPFMTFSDYLEGMYEADAKIERESVSELLASGDDLFKKIDEIRNNYSGVEFDVEKFQEFCKELNVNPMLIGHVIEAIFSQKAGVTVTGLGTLSPEMGASVALSNTSVDTCEDMDVTEDMEDIVLMADKSHSYMSPEMARWADVKYDNNKGGLVEYKVGTSMTLPATWAEKGKAVLPLSGICVRKPQFSKPLDEEDDLRLSNMNFFKVSDLKLKKTITPVVYTGTIRERQMKNYIDYLSASLGSTLGNLERIVRSDWNGTEESMQTFGLYDCEKCKWLLLPAEKKHAWAVVLASDDTTRIIFLSYDESGSPIIDKRNWKRFAVCSETKVYSVIRSLEVLNKEAIVDPGVHITLVDGVPGCGKTAEIIARVNWKTDLVLTPGREAAAMIRRRACALHKSPVATSDNVRTFDSFVMNKKIFKFDAVYVDEGLMVHTGLLNFALKISGCKKAFVFGDAKQIPFINRVMNFDYPKELRTLIVDNVERRYVTHRCPRDVTSFLNTIYKAAVATTSPVVHSVKAIKVSGAGILRPELTKIKGKIITFTQSDKQSLIKSGYNDVNTVHEIQGETFEETAVVRATPTPIGLIVRDSPHVLVALTRHTKAMVYYTVVFDAVTSIIADVEKVDQSILTMFATTVPTKXQLMQNSLYVHRNIFLPVSKTGFYTDMQEFYDRCLPGNSFVLNDFDAVTMRLRDNEFNLQPCRLTLSNLDPVPALVKSEAQNFLIPVLRTACERPRIPGLLENLVAMIKRNMNTPDLAGTVDITNMSISIVDNFFSSFVRDEVLLDHLDCVRASSIQSFSDWFSCQPTSAVGQLANFNFIDLPAFDTYMHMIKRQPKSRLDTSIQSEYPALQTIVYHPKVVNAVFGPVFKYLTTKFLSMVDSSKFFFYTRKKPEDLQEFFSDLSSHSDYEILELDVSKYDKSQSDFHFSIEMAIWEKLGLDDILAWMWSMGHKRTILQDFQAGIKTLIYYQRKSGDVTTFIGNTFIIAACVASMLPLDKCFKASFCGDDSLIYLPKGLEYPDIQATANLVWNFEAKLFRKKYGYFCGKYIIHHANGCIVYPDPLKLISKLGNKSLVGYEHVEEFRISLLDVAHSLFNGAYFHLLDDAIHELFPNAGGCSFVINCLCKYLSDKRLFRSLYIDVSK.

The 186 kDa protein encoded by the attenuated CGMMV strain ONAL-1differs from the 186 kDa protein encoded by the wild type CGMMV Ontariostrain (SEQ ID NO:64) at least in that position 1092 of SEQ ID NO:55 isvaline (V, Val).

Mutant CGMMV Ontario Strain ONAL-2

Directed mutation of the cDNA genome of the cloned CGMMV Ontario strain(Example 1) was carried out as described above, using the mutagenicprimers listed in Table 6 to introduce the mutation c.4969G>A.Nucleotide residues indicated in bold indicate sites of mutation. Thismutation resulted in an R1637H amino acid substitution in the encodedviral 186 kDa protein. The resulting mutant CGMMV strain was designatedOntario strain ONAL-2.

TABLE 6 Primers used to produce mutant CGMMV Ontario strain ONAL-2Sequence Primer Sequence (5′ to 3′) IdentifierCTATATAAAGACTACGGAAAAGGTGCTTATCACTCAAAT SEQ ID ACTTGCAC NO: 56GTGCAAGTATTTGAGTGATAAGCACCTTTTCCGTAGTCT SEQ ID TTATATAG NO: 57

The cDNA genome sequence of CGMMV strain ONAL-2 is shown below.

(SEQ ID NO: 58) GTTTTAATTTTTAAAATTAAACAAACAACAACAACAACAACAAACAATTTAAAACAACAATGGCAAACATTAATGAACAAATCAACAACCAACGCGACGCCGCGGCCAGCGGGAGAAACAATCTCGTTAGCCAATTGGCGTCAAAAAGGGTGTATGACGAGGCTGTTCGCTCGTTGGATCATCAAGACAGACGCCCAAAAATGAACTTTTCTCGTGTGGTCAGCACAGAGCACACCAGGCTTGTAACTGATGCGTATCCGGAGTTTTCGATTAGCTTTACCGCCACCAAGAACTCTGTACACTCCCTTGCGGGTGGTCTGAGGCTCCTTGAACTGGAATATATGATGATGCAAGTGCCCTACGGCTCACCTTGTTATGATATCGGCGGTAACTATACGCAGCACTTGTTCAAAGGTAGATCATATGTGCATTGCTGCAATCCGTGCCTGGATCTTAAAGATGTTGCGAGGAACGTGATGTATAACGATATGGTCACACAACATGTACAGAGGCACAAGGGATCTGGCGGGTGCAGACCTCTTCCAACTTTTCAGATAGATGCATTCAGGAGGTACGATAATTCTCCCTGTGCGGTCACCTGTTCAGACGTTTTCCAAGAGTGTTCCTATGATTTTGGGAGCGGTAGGGATAATCATGCAGTCTCGCTGCATTCAATCTACGATATCCCTTATTCTTCGATCGGACCTGCTCTTCATAGGAAGAACGTGCGAGTTTGTTATGCAGCCTTTCACTTCTCGGAGGCATTGCTTTTAGGTTCACCTGTAGGTAATTTAAATAGTATTGGGGCTCAGTTTAGGGTCGATGGTGATGATGTGCATTTTCTTTTTAGTGAAGAGTCTACTTTGCATTATACTCATAGTTTAGAAAATATCAAATTAATTGTGATGCGTACTTATTTTCCTGCTGATGATAGGTACGTGTATATTAAGGAGTTTATGGTCAAGCGTGTGGATACTTTCTTCTTTAGGTTGGTCAGAGCAGACACACATATGCTTCATAAATCTGTGGGGCACTATTCAAAATCGAAATCTGAGTACTTTGCGCTGAATACCCCTCCGATCTTCCAAGACAAAGCCACGTTTTCTGTGTGGTTTCCTGAGGCGAAGCGTAAGGTGTTGATACCCAAGTTTGAACTTTCAAGATTCCTTTCTGGGAATGTGAAAATCTCTAGGATGCTTGTCGATGCTGATTTCGTCCATACCATTATTAATCACATTAGCACGTATGATAATAAGGCCTTAGTGTGGAAGAATGTTCAGTCCTTTGTGGAATCTATACGCTCAAGAGTAATTGTAAACGGAGTTTCGGTGAAATCTGAATGGAACGTACCGGTTGATCAGCTCACTGATATCTCGTTCTCGATATTCCTTCTCGTGAAGGTTAGGAAGGTACAGATCGAGTTAATGTCTGATAAAGTTGTAATCGAGGCGAGGGGCTTGCTCCGGAGGTTCGCAGACAGTCTTAAATCCGCCGTAGAAGGACTAGGTGATTGCGTCTATGATGCTCTAGTTCAAACCGGCTGGTTTGATACCTCTAGCGACGAACTGAAAGTTTTGCTACCTGAACCGTTTATGACCTTTTCGGATTATCTTGAAGGGATGTACGAGGCAGATGCAAAGATCGAGAGAGAGAGTGTCTCTGAGTTGCTCGCTTCCGGTGACGATTTGTTCAAGAAAATCGATGAGATAAGAAACAATTACAGTGGAGTCGAATTTGATGTAGAGAAATTCCAGGAATTTTGCAAGGAACTGAATGTTAATCCTATGCTAATTGGCCATGTTATCGAAGCTATTTTTTCGCAGAAAGCTGGGGTGACAGTAACGGGTCTGGGTACCCTCTCTCCTGAGATGGGTGCTTCTGTTGCGTTATCCAATACCTCTGTAGATACATGTGAAGATATGGATGTAACTGAAGATATGGAGGATATAGTGTTGATGGCGGACAAGAGTCATTCTTACATGTCCCCAGAAATGGCGAGATGGGCTGATGTAAAATACGACAACAATAAAGGGGGCCTGGTCGAATACAAAGTCGGAACCTCGATGACTTTACCTGCCACCTGGGCAGAGAAGGGTAAGGCTGTCTTACCGTTGTCGGGGATCTGTGTGAGGAAACCCCAATTTTCGAAGCCGCTTGATGAGGAAGACGACTTGAGGTTATCAAACATGAATTTCTTTAAGGTGAGCGATCTGAAGTTGAAGAAAACTATCACTCCAGTTGTTTACACTGGGACCATTCGAGAGAGGCAAATGAAGAATTATATTGATTACTTATCGGCCTCTCTTGGTTCTACGCTGGGTAATCTGGAGAGAATTGTGCGGAGTGATTGGAACGGTACCGAGGAGAGTATGCAAACGTTCGGGTTGTATGACTGCGAAAAGTGCAAGTGGTTACTGTTACCAGCCGAAAAGAAGCACGCATGGGCTGTGGTTCTGGCAAGTGATGATACCACTCGCATAATCTTCCTCTCATATGACGAATCTGGTTCTCCCATAATTGATAAGAGAAACTGGAAGCGATTTGCTGTTTGCTCTGAGACCAAAGTCTATAGCGTAATTCGTAGTTTAGAGGTACTAAATAAGGAAGCAATAGTCGACCCCGGGGTTCATATAACATTAGTTGACGGAGTGCCGGGTTGTGGAAAGACCGCCGAAATTATAGCGAGGGTCAATTGGAAAACCGATCTAGTATTGACTCCCGGGAGGGAGGCGGCTGCTATGATTAGGCGGAGGGCCTGCGCCCTGCACAAGTCACCTGTGGCAACCAGTGACAACGTTAGAACTTTCGATTCTTTTGTGATGAATAAGAAAATCTTCAAGTTTGACGCTGTCTATGTTGACGAGGGTCTGATGGTCCATACGGGTTTACTTAATTTTGCGTTGAAGATCTCAGGTTGTAAAAAGGCCTTCGTCTTTGGTGATGCTAAGCAAATCCCGTTTATAAACAGAGTCATGAATTTTGATTATCCTAAGGAGTTAAGAACTTTAATAGTCGATAATGTAGAGCGTAGGTATGTTACCCATAGGTGTCCTAGAGATGTCACTAGTTTTCTTAATACTATTTACAAAGCCGCTGTCGCTACTACTAGTCCGGTTGTACATTCTGTGAAGGCGATTAAAGTGTCAGGGGCCGGTATTCTGAGGCCCGAGTTGACGAAGATCAAAGGAAAGATAATAACGTTTACTCAATCTGATAAGCAGTCCTTGATCAAGAGTGGGTACAATGACGTGAACACTGTGCATGAAATTCAGGGAGAAACCTTTGAAGAGACGGCGGTTGTGCGTGCCACCCCGACTCCGATAGGTTTAATTGCCCGTGATTCACCACATGTACTAGTGGCCTTAACGAGGCACACTAAGGCAATGGTGTATTATACTGTTGTGTTCGATGCAGTTACAAGTATAATAGCGGATGTGGAAAAGGTCGACCAGTCGATCTTGACTATGTTTGCTACCACTGTGCCTACCAAATAGCAATTAATGCAGAACTCACTGTATGTCCATCGTAATATTTTCCTCCCTGTTAGTAAAACGGGGTTTTATACAGACATGCAGGAGTTCTATGATAGATGCCTTCCTGGGAATTCCTTCGTGCTGAATGATTTCGATGCCGTAACCATGCGGTTGAGGGACAACGAATTTAACCTACAACCTTGTAGGCTAACCTTAAGTAATTTAGATCCAGTACCCGCTTTGGTTAAGAGTGAAGCGCAGAATTTTCTGATTCCCGTTTTGCGTACGGCCTGTGAAAGGCCGCGCATTCCAGGTCTCCTTGAAAATCTTGTAGCTATGATAAAGAGGAATATGAATACTCCTGATCTAGCTGGGACTGTGGATATAACTAATATGTCGATTTCTATAGTAGATAACTTCTTTTCTTCTTTTGTTAGAGACGAGGTTTTGCTTGATCATTTAGATTGTGTTAGGGCTAGTTCCATTCAAAGTTTTTCTGATTGGTTTTCGTGTCAGCCAACCTCGGCGGTTGGTCAATTAGCTAATTTCAATTTCATAGATTTGCCTGCCTTTGATACTTATATGCACATGATTAAGCGGCAGCCCAAGAGTCGGTTGGATACTTCGATTCAGTCTGAATATCCGGCCTTGCAAACTATTGTTTATCACCCTAAAGTGGTAAATGCAGTTTTCGGTCCGGTTTTTAAGTATTTGACCACCAAGTTTCTTAGCATGGTAGATAGTTCTAAGTTTTTCTTTTACACTAGGAAAAAACCAGAAGATCTGCAGGAATTTTTCTCAGATCTCTCTTCCCATTCTGATTATGAGATTCTTGAGCTGGATGTTTCTAAATATGACAAGTCACAATCCGATTTCCATTTCTCTATTGAGATGGCAATTTGGGAAAAATTGGGGCTGGACGATATTTTGGCTTGGATGTGGTCTATGGGTCACAAGAGAACTATACTGCAAGATTTCCAAGCCGGGATAAAGACGCTCATTTACTATCAACGGAAGTCTGGTGATGTAACTACTTTCATAGGTAATACCTTTATTATCGCAGCGTGTGTAGCTAGTATGTTGCCGTTAGACAAGTGTTTTAAAGCTAGTTTTTGTGGTGATGATTCGCTGATCTACCTTCCTAAGGGTTTGGAGTATCCTGATATACAGGCTACTGCCAACTTGGTTTGGAATTTTGAGGCGAAACTTTTCCGAAAGAAGTATGGTTACTTCTGTGGGAAGTATATAATTCACCATGCCAACGGCTGTATTGTTTACCCTGACCCTTTAAAATTAATTAGTAAATTAGGTAATAAGAGTCTTGTAGGGTATGAGCATGTTGAGGAGTTTCGTATATCTCTCCTCGACGTCGCTCATAGTTTGTTTAATGGTGCTTATTTCCATTTACTCGACGATGCAATCCACGAATTATTTCCTAACGCTGGGGGTTGCAGTTTTGTAATTAATTGTTTGTGCAAGTATTTGAGTGATAAGCACCTTTTCCGTAGTCTTTATATAGATGTCTCTAAGTAAGGTGTCGGTCGAGAACTCATTGAAACCCGAGAAGTTTGTTAAAATCTCTTGGGTCGATAAGTTGCTCCCTAACTATTTTTCCATTCTTAAGTATTTATCTATAACTGACTTTAGCGTAGTTAAAGCTCAGAGCTATGAATCCCTCGTGCCTGTCAAGTTGTTGCGTGGTGTTGATCTTACAAAACACCTTTATGTCACATTGTTGGGCGTTGTGGTTTCTGGTGTATGGAACGTACCGGAATCCTGTAGGGGTGGTGCTACTGTTGCTCTGGTTGACACAAGGATGCATTCTGTTGCAGAGGGAACTATATGCAAATTTTCAGCTCCCGCCACCGTCCGCGAATTCTCTGTTAGGTTCATACCTAACTATTCTGTCGTGGCTGCGGATGCCCTTCGCGATCCTTGGTCTTTATTTGTGAGACTCTCTAATGTAGGGATTAAAGATGGTTTCCATCCTTTGACCTTAGAGGTCGCTTGTTTAGTCGCTACAACTAACTCTATTATCAAAAAGGGTCTTAGAGCTTCTGTAGTCGAGTCTGTCGTCTCTTCCGATCAGTCCATTGTCCTAGATTCTTTATCCGAGAAAGTTGAACCTTTCTTTGATAAAGTTCCTATTTCGGCGGCTGTGATGGCAAGAGACCCCAGTTATAGGTCTAGGTCGCAGTCTGTCGGTGGTCGTGGTAAGCGGCATTCTAAACCTCCAAATCGGAGGTTGGACTCTGCTTCTGAAGAGTCCAGTTCTGTTTCTTTCGAAGATGGCTTACAATCCGATCACACCTAGCAAACTTATTGCGTTTAGTGCTTCTTATGTTCCCGTCAGGACTTTACTTAATTTTCTAGTTGCTTCACAAGGTACCGCCTTCCAGACTCAAGCGGGAAGAGATTCTTTCCGCGAGTCCCTGTCTGCGTTACCCTCGTCTGTCGTAGATATTAATTCTAGGTTCCCAAATGCGGGTTTTTACGCTTTCCTCAACGGTCCTGTGTTGAGGCCTATCTTCGTTTCGCTTCTTAGCTCTACGGATACGCGTAATAGGGTCATTGAGGTTGTAGATCCTAGCAATCCTACGACTGCTGAGTCGCTTAACGCTGTAAAGCGTACTGATGACGCATCTACGGCCGCTAGGGCTGAAATAGATAATTTAATAGAGTCTATTTCTAAGGGTTTTGATGTTTATGATAGGGCTTCATTTGAAGCCGCGTTTTCGGTAGTCTGGTCAGAGGCTACCACCTCGAAAGCTTAGCTTCGAGGGTCTTCTGATGGTGGTGCACACCAAAGTGCATAGTGCTTTCCCGTTCACTTAAATCGAACGGTTTGCTCATTGGTTTGCGGAAACCTCTCACGTGTGGCGTTGAAGTTTCTATGGGCAGTAATTCTGCAAGGGGTTCGAATCCCCCCTTTCCCCGGGTAGGGGCCCA.

The cDNA genome sequence of the attenuated CGMMV strain ONAL-2 differsfrom the cDNA genome sequence of the wild type CGMMV Ontario strain (SEQID NO:18) at least in that the nucleotide at position 4969 of SEQ IDNO:58 is A.

The 186 kDa protein encoded by the attenuated CGMMV strain ONAL-2 hasthe following sequence.

(SEQ ID NO: 59) MANINEQINNQRDAAASGRNNLVSQLASKRVYDEAVRSLDHQDRRPKMNFSRVVSTEHTRLVTDAYPEFSISFTATKNSVHSLAGGLRLLELEYMMMQVPYGSPCYDIGGNYTQIILFKGRSYVHCCNPCLDLKDVARNVMYNDMVTQHVQRHKGSGGCRPLPTFQIDAFRRYDNSPCAVTCSDVFQECSYDFGSGRDNHAVSLHSIYDIPYSSIGPALHRKNVRVCYAAFHFSEALLLGSPVGNLNSIGAQFRVDGDDVHFLFSEESTLHYTHSLENIKLIVMRTYFPADDRYVYIKEFMVKRVDTFFFRLVRADTHMLHKSVGHYSKSKSEYFALNTPPIFQDKATFSVWFPEAKRKVLIPKFELSRFLSGNVKISRMLVDADFVHTIINHISTYDNKALVWKNVQSFVESIRSRVIVNGVSVKSEWNVPVDQLTDISFSIFLLVKVRKVQIELMSDKVVIEARGLLRRFADSLKSAVEGLGDCVYDALVQTGWFDTSSDELKVLLPEPFMTFSDYLEGMYEADAKIERESVSELLASGDDLFKKIDEIRNNYSGVEFDVEKFQEFCKELNVNPMLIGHVIEAIFSQKAGVTVTGLGTLSPEMGASVALSNTSVDTCEDMDVTEDMEDIVLMADKSHSYMSPEMARWADVKYDNNKGGLVEYKVGTSMTLPATWAEKGKAVLPLSGICVRKPQFSKPLDEEDDLRLSNMNFFKVSDLKLKKTITPVVYTGTIRERQMKNYIDYLSASLGSTLGNLERIVRSDWNGTEESMQTFGLYDCEKCKWLLLPAEKKHAWAVVLASDDTTRIIFLSYDESGSPIIDKRNWKRFAVCSETKVYSVIRSLEVLNKEAIVDPGVHITLVDGVPGCGKTAEIIARVNWKTDLVLTPGREAAAMIRRRACALHKSPVATSDNVRTFDSFVMNKKIFKFDAVYVDEGLMVHTGLLNFALKISGCKKAFVFGDAKQIPFINRVMNFDYPKELRTLIVDNVERRYVTHRCPRDVTSFLNTIYKAAVATTSPVVHSVKAIKVSGAGILRPELTKIKGKIITFTQSDKQSLIKSGYNDVNTVHEIQGETFEETAVVRATPTPIGLIARDSPHVLVALTRHTKAMVYYTVVFDAVTSIIADVEKVDQSILTMFATTVPTKXQLMQNSLYVHRNIFLPVSKTGFYTDMQEFYDRCLPGNSFVLNDFDAVTMRLRDNEFNLQPCRLTLSNLDPVPALVKSEAQNFLIPVLRTACERPRIPGLLENLVAMIKRNMNTPDLAGTVDITNMSISIVDNFFSSFVRDEVLLDHLDCVRASSIQSFSDWFSCQPTSAVGQLANFNFIDLPAFDTYMHMIKRQPKSRLDTSIQSEYPALQTIVYHPKVVNAVFGPVFKYLTTKFLSMVDSSKFFFYTRKKPEDLQEFFSDLSSHSDYEILELDVSKYDKSQSDFHFSIEMAIWEKLGLDDILAWMWSMGHKRTILQDFQAGIKTLIYYQRKSGDVTTFIGNTFIIAACVASMLPLDKCFKASFCGDDSLIYLPKGLEYPDIQATANLVWNFEAKLFRKKYGYFCGKYIIHHANGCIVYPDPLKLISKLGNKSLVGYEHVEEFRISLLDVAHSLFNGAYFHLLDDAIHELFPNAGGCSFVINCLCKYLSDKHLFRSLYIDVSK.

The 186 kDa protein encoded by the attenuated CGMMV strain ONAL-2differs from the 186 kDa protein encoded by the wild type CGMMV Ontariostrain (SEQ ID NO:64) at least in that position 1637 of SEQ ID NO:59 ishistidine (H, His).

Mutant CGMMV Ontario Strain ONBM-32

Directed mutation of the cDNA genome of the cloned CGMMV Ontario strain(Example 1) was carried out as described above to introduce mutationscorresponding to those induced in the cDNA genome of the cloned CGMMVOntario strain mutants ONBM, ONAL-1 and ONAL-2 (c.315G>A; c.1498A>G;c.1660C>T; c.3334C>T c.3430C>T; c.3528A>G; c.4144C>T; c.4248C>T;c.4969G>A; and c.6228C>T). These mutations resulted in amino acidsubstitutions in the encoded viral proteins (G86S, E480G, S534F, A1092Vand A1124V in the 129 kDa protein; G86S, E480G, S534F, A1092V, A1124V,N1157D, P1362L, P1397S and R1637H in the 186 kDa protein; and A156V inthe coat protein). The resulting mutant CGMMV strain was designatedOntario strain ONBM-32.

The cDNA genome sequence of CGMMV strain ONBM-32 is shown below.

(SEQ ID NO: 60) GTTTTAATTTTTAAAATTAAACAAACAACAACAACAACAACAAACAATTTAAAACAACAATGGCAAACATTAATGAACAAATCAACAACCAACGCGACGCCGCGGCCAGCGGGAGAAACAATCTCGTTAGCCAATTGGCGTCAAAAAGGGTGTATGACGAGGCTGTTCGCTCGTTGGATCATCAAGACAGACGCCCAAAAATGAACTTTTCTCGTGTGGTCAGCACAGAGCACACCAGGCTTGTAACTGATGCGTATCCGGAGTTTTCGATTAGCTTTACCGCCACCAAGAACTCTGTACACTCCCTTGCGGGTAGTCTGAGGCTCCTTGAACTGGAATATATGATGATGCAAGTGCCCTACGGCTCACCTTGTTATGATATCGGCGGTAACTATACGCAGCACTTGTTCAAAGGTAGATCATATGTGCATTGCTGCAATCCGTGCCTGGATCTTAAAGATGTTGCGAGGAACGTGATGTATAACGATATGGTCACACAACATGTACAGAGGCACAAGGGATCTGGCGGGTGCAGACCTCTTCCAACTTTTCAGATAGATGCATTCAGGAGGTACGATAATTCTCCCTGTGCGGTCACCTGTTCAGACGTTTTCCAAGAGTGTTCCTATGATTTTGGGAGCGGTAGGGATAATCATGCAGTCTCGCTGCATTCAATCTACGATATCCCTTATTCTTCGATCGGACCTGCTCTTCATAGGAAGAACGTGCGAGTTTGTTATGCAGCCTTTCACTTCTCGGAGGCATTGCTTTTAGGTTCACCTGTAGGTAATTTAAATAGTATTGGGGCTCAGTTTAGGGTCGATGGTGATGATGTGCATTTTCTTTTTAGTGAAGAGTCTACTTTGCATTATACTCATAGTTTAGAAAATATCAAATTAATTGTGATGCGTACTTATTTTCCTGCTGATGATAGGTACGTGTATATTAAGGAGTTTATGGTCAAGCGTGTGGATACTTTCTTCTTTAGGTTGGTCAGAGCAGACACACATATGCTTCATAAATCTGTGGGGCACTATTCAAAATCGAAATCTGAGTACTTTGCGCTGAATACCCCTCCGATCTTCCAAGACAAAGCCACGTTTTCTGTGTGGTTTCCTGAGGCGAAGCGTAAGGTGTTGATACCCAAGTTTGAACTTTCAAGATTCCTTTCTGGGAATGTGAAAATCTCTAGGATGCTTGTCGATGCTGATTTCGTCCATACCATTATTAATCACATTAGCACGTATGATAATAAGGCCTTAGTGTGGAAGAATGTTCAGTCCTTTGTGGAATCTATACGCTCAAGAGTAATTGTAAACGGAGTTTCGGTGAAATCTGAATGGAACGTACCGGTTGATCAGCTCACTGATATCTCGTTCTCGATATTCCTTCTCGTGAAGGTTAGGAAGGTACAGATCGAGTTAATGTCTGATAAAGTTGTAATCGAGGCGAGGGGCTTGCTCCGGAGGTTCGCAGACAGTCTTAAATCCGCCGTAGGAGGACTAGGTGATTGCGTCTATGATGCTCTAGTTCAAACCGGCTGGTTTGATACCTCTAGCGACGAACTGAAAGTTTTGCTACCTGAACCGTTTATGACCTTTTCGGATTATCTTGAAGGGATGTACGAGGCAGATGCAAAGATCGAGAGAGAGAGTGTCTTTGAGTTGCTCGCTTCCGGTGACGATTTGTTCAAGAAAATCGATGAGATAAGAAACAATTACAGTGGAGTCGAATTTGATGTAGAGAAATTCCAGGAATTTTGCAAGGAACTGAATGTTAATCCTATGCTAATTGGCCATGTTATCGAAGCTATTTTTTCGCAGAAAGCTGGGGTGACAGTAACGGGTCTGGGTACCCTCTCTCCTGAGATGGGTGCTTCTGTTGCGTTATCCAATACCTCTGTAGATACATGTGAAGATATGGATGTAACTGAAGATATGGAGGATATAGTGTTGATGGCGGACAAGAGTCATTCTTACATGTCCCCAGAAATGGCGAGATGGGCTGATGTAAAATACGACAACAATAAAGGGGGCCTGGTCGAATACAAAGTCGGAACCTCGATGACTTTACCTGCCACCTGGGCAGAGAAGGGTAAGGCTGTCTTACCGTTGTCGGGGATCTGTGTGAGGAAACCCCAATTTTCGAAGCCGCTTGATGAGGAAGACGACTTGAGGTTATCAAACATGAATTTCTTTAAGGTGAGCGATCTGAAGTTGAAGAAAACTATCACTCCAGTTGTTTACACTGGGACCATTCGAGAGAGGCAAATGAAGAATTATATTGATTACTTATCGGCCTCTCTTGGTTCTACGCTGGGTAATCTGGAGAGAATTGTGCGGAGTGATTGGAACGGTACCGAGGAGAGTATGCAAACGTTCGGGTTGTATGACTGCGAAAAGTGCAAGTGGTTACTGTTACCAGCCGAAAAGAAGCACGCATGGGCTGTGGTTCTGGCAAGTGATGATACCACTCGCATAATCTTCCTCTCATATGACGAATCTGGTTCTCCCATAATTGATAAGAGAAACTGGAAGCGATTTGCTGTTTGCTCTGAGACCAAAGTCTATAGCGTAATTCGTAGTTTAGAGGTACTAAATAAGGAAGCAATAGTCGACCCCGGGGTTCATATAACATTAGTTGACGGAGTGCCGGGTTGTGGAAAGACCGCCGAAATTATAGCGAGGGTCAATTGGAAAACCGATCTAGTATTGACTCCCGGGAGGGAGGCGGCTGCTATGATTAGGCGGAGGGCCTGCGCCCTGCACAAGTCACCTGTGGCAACCAGTGACAACGTTAGAACTTTCGATTCTTTTGTGATGAATAAGAAAATCTTCAAGTTTGACGCTGTCTATGTTGACGAGGGTCTGATGGTCCATACGGGTTTACTTAATTTTGCGTTGAAGATCTCAGGTTGTAAAAAGGCCTTCGTCTTTGGTGATGCTAAGCAAATCCCGTTTATAAACAGAGTCATGAATTTTGATTATCCTAAGGAGTTAAGAACTTTAATAGTCGATAATGTAGAGCGTAGGTATGTTACCCATAGGTGTCCTAGAGATGTCACTAGTTTTCTTAATACTATTTACAAAGCCGCTGTCGCTACTACTAGTCCGGTTGTACATTCTGTGAAGGCGATTAAAGTGTCAGGGGCCGGTATTCTGAGGCCCGAGTTGACGAAGATCAAAGGAAAGATAATAACGTTTACTCAATCTGATAAGCAGTCCTTGATCAAGAGTGGGTACAATGACGTGAACACTGTGCATGAAATTCAGGGAGAAACCTTTGAAGAGACGGCGGTTGTGCGTGCCACCCCGACTCCGATAGGTTTAATTGTCCGTGATTCACCACATGTACTAGTGGCCTTAACGAGGCACACTAAGGCAATGGTGTATTATACTGTTGTGTTCGATGCAGTTACAAGTATAATAGTGGATGTGGAAAAGGTCGACCAGTCGATCTTGACTATGTTTGCTACCACTGTGCCTACCAAATAGCAATTAATGCAGAACTCACTGTATGTCCATCGTGATATTTTCCTCCCTGTTAGTAAAACGGGGTTTTATACAGACATGCAGGAGTTCTATGATAGATGCCTTCCTGGGAATTCCTTCGTGCTGAATGATTTCGATGCCGTAACCATGCGGTTGAGGGACAACGAATTTAACCTACAACCTTGTAGGCTAACCTTAAGTAATTTAGATCCAGTACCCGCTTTGGTTAAGAGTGAAGCGCAGAATTTTCTGATTCCCGTTTTGCGTACGGCCTGTGAAAGGCCGCGCATTCCAGGTCTCCTTGAAAATCTTGTAGCTATGATAAAGAGGAATATGAATACTCCTGATCTAGCTGGGACTGTGGATATAACTAATATGTCGATTTCTATAGTAGATAACTTCTTTTCTTCTTTTGTTAGAGACGAGGTTTTGCTTGATCATTTAGATTGTGTTAGGGCTAGTTCCATTCAAAGTTTTTCTGATTGGTTTTCGTGTCAGCCAACCTCGGCGGTTGGTCAATTAGCTAATTTCAATTTCATAGATTTGCCTGCCTTTGATACTTATATGCACATGATTAAGCGGCAGCCCAAGAGTCGGTTGGATACTTCGATTCAGTCTGAATATCCGGCCTTGCAAACTATTGTTTATCACCTTAAAGTGGTAAATGCAGTTTTCGGTCCGGTTTTTAAGTATTTGACCACCAAGTTTCTTAGCATGGTAGATAGTTCTAAGTTTTTCTTTTACACTAGGAAAAAATCAGAAGATCTGCAGGAATTTTTCTCAGATCTCTCTTCCCATTCTGATTATGAGATTCTTGAGCTGGATGTTTCTAAATATGACAAGTCACAATCCGATTTCCATTTCTCTATTGAGATGGCAATTTGGGAAAAATTGGGGCTGGACGATATTTTGGCTTGGATGTGGTCTATGGGTCACAAGAGAACTATACTGCAAGATTTCCAAGCCGGGATAAAGACGCTCATTTACTATCAACGGAAGTCTGGTGATGTAACTACTTTCATAGGTAATACCTTTATTATCGCAGCGTGTGTAGCTAGTATGTTGCCGTTAGACAAGTGTTTTAAAGCTAGTTTTTGTGGTGATGATTCGCTGATCTACCTTCCTAAGGGTTTGGAGTATCCTGATATACAGGCTACTGCCAACTTGGTTTGGAATTTTGAGGCGAAACTTTTCCGAAAGAAGTATGGTTACTTCTGTGGGAAGTATATAATTCACCATGCCAACGGCTGTATTGTTTACCCTGACCCTTTAAAATTAATTAGTAAATTAGGTAATAAGAGTCTTGTAGGGTATGAGCATGTTGAGGAGTTTCGTATATCTCTCCTCGACGTCGCTCATAGTTTGTTTAATGGTGCTTATTTCCATTTACTCGACGATGCAATCCACGAATTATTTCCTAACGCTGGGGGTTGCAGTTTTGTAATTAATTGTTTGTGCAAGTATTTGAGTGATAAGCACCTTTTCCGTAGTCTTTATATAGATGTCTCTAAGTAAGGTGTCGGTCGAGAACTCATTGAAACCCGAGAAGTTTGTTAAAATCTCTTGGGTCGATAAGTTGCTCCCTAACTATTTTTCCATTCTTAAGTATTTATCTATAACTGACTTTAGCGTAGTTAAAGCTCAGAGCTATGAATCCCTCGTGCCTGTCAAGTTGTTGCGTGGTGTTGATCTTACAAAACACCTTTATGTCACATTGTTGGGCGTTGTGGTTTCTGGTGTATGGAACGTACCGGAATCCTGTAGGGGTGGTGCTACTGTTGCTCTGGTTGACACAAGGATGCATTCTGTTGCAGAGGGAACTATATGCAAATTTTCAGCTCCCGCCACCGTCCGCGAATTCTCTGTTAGGTTCATACCTAACTATTCTGTCGTGGCTGCGGATGCCCTTCGCGATCCTTGGTCTTTATTTGTGAGACTCTCTAATGTAGGGATTAAAGATGGTTTCCATCCTTTGACCTTAGAGGTCGCTTGTTTAGTCGCTACAACTAACTCTATTATCAAAAAGGGTCTTAGAGCTTCTGTAGTCGAGTCTGTCGTCTCTTCCGATCAGTCCATTGTCCTAGATTCTTTATCCGAGAAAGTTGAACCTTTCTTTGATAAAGTTCCTATTTCGGCGGCTGTGATGGCAAGAGACCCCAGTTATAGGTCTAGGTCGCAGTCTGTCGGTGGTCGTGGTAAGCGGCATTCTAAACCTCCAAATCGGAGGTTGGACTCTGCTTCTGAAGAGTCCAGTTCTGTTTCTTTCGAAGATGGCTTACAATCCGATCACACCTAGCAAACTTATTGCGTTTAGTGCTTCTTATGTTCCCGTCAGGACTTTACTTAATTTTCTAGTTGCTTCACAAGGTACCGCCTTCCAGACTCAAGCGGGAAGAGATTCTTTCCGCGAGTCCCTGTCTGCGTTACCCTCGTCTGTCGTAGATATTAATTCTAGGTTCCCAAATGCGGGTTTTTACGCTTTCCTCAACGGTCCTGTGTTGAGGCCTATCTTCGTTTCGCTTCTTAGCTCTACGGATACGCGTAATAGGGTCATTGAGGTTGTAGATCCTAGCAATCCTACGACTGCTGAGTCGCTTAACGCTGTAAAGCGTACTGATGACGCATCTACGGCCGCTAGGGCTGAAATAGATAATTTAATAGAGTCTATTTCTAAGGGTTTTGATGTTTATGATAGGGCTTCATTTGAAGCCGCGTTTTCGGTAGTCTGGTCAGAGGTTACCACCTCGAAAGCTTAGCTTCGAGGGTCTTCTGATGGTGGTGCACACCAAAGTGCATAGTGCTTTCCCGTTCACTTAAATCGAACGGTTTGCTCATTGGTTTGCGGAAACCTCTCACGTGTGGCGTTGAAGTTTCTATGGGCAGTAATTCTGCAAGGGGTTCGAATCCCCCCTTTCCCCGGGTAGGGGCCCA.

The cDNA genome sequence of the attenuated CGMMV strain ONBM-32 differsfrom the cDNA genome sequence of the wild type CGMMV Ontario strain (SEQID NO:18) at least in that:

the nucleotide at position 315 of SEQ ID NO:60 is A;

the nucleotide at position 1498 of SEQ ID NO:60 is G;

the nucleotide at position 1660 of SEQ ID NO:60 is T;

the nucleotide at position 3334 of SEQ ID NO:60 is T;

the nucleotide at position 3430 of SEQ ID NO:60 is T;

the nucleotide at position 3528 of SEQ ID NO:60 is G;

the nucleotide at position 4144 of SEQ ID NO:60 is T;

the nucleotide at position 4248 of SEQ ID NO:60 is T;

the nucleotide at position 4969 of SEQ ID NO:60 is A; and

the nucleotide at position 6228 of SEQ ID NO:60 is T.

The 129 kDa protein encoded by the attenuated CGMMV strain ONBM-32 hasthe following sequence.

(SEQ ID NO: 61) MANINEQINNQRDAAASGRNNLVSQLASKRVYDEAVRSLDHQDRRPKMNFSRVVSTEHTRLVTDAYPEFSISFTATKNSVHSLAGSLRLLELEYMMMQVPYGSPCYDIGGNYTQHLFKGRSYVHCCNPCLDLKDVARNVMYNDMVTQHVQRHKGSGGCRPLPTFQIDAFRRYDNSPCAVTCSDVFQECSYDFGSGRDNHAVSLHSIYDIPYSSIGPALHRKNVRVCYAAFHFSEALLLGSPVGNLNSIGAQFRVDGDDVHFLFSEESTLHYTHSLENIKLIVMRTYFPADDRYVYIKEFMVKRVDTFFFRLVRADTHMLHKSVGHYSKSKSEYFALNTPPIFQDKATFSVWFPEAKRKVLIPKFELSRFLSGNVKISRMLVDADFVHTIINHISTYDNKALVWKNVQSFVESIRSRVIVNGVSVKSEWNVPVDQLTDISFSIFLLVKVRKVQIELMSDKVVIEARGLLRRFADSLKSAVGGLGDCVYDALVQTGWFDTSSDELKVLLPEPFMTFSDYLEGMYEADAKIERESVFELLASGDDLFKKIDEIRNNYSGVEFDVEKFQEFCKELNVNPMLIGHVIEAIFSQKAGVTVTGLGTLSPEMGASVALSNTSVDTCEDMDVTEDMEDIVLMADKSHSYMSPEMARWADVKYDNNKGGLVEYKVGTSMTLPATWAEKGKAVLPLSGICVRKPQFSKPLDEEDDLRLSNMNFFKVSDLKLKKTITPVVYTGTIRERQMKNYIDYLSASLGSTLGNLERIVRSDWNGTEESMQTFGLYDCEKCKWLLLPAEKKHAWAVVLASDDTTRIIFLSYDESGSPIIDKRNWKRFAVCSETKVYSVIRSLEVLNKEAIVDPGVHITLVDGVPGCGKTAEIIARVNWKTDLVLTPGREAAAMIRRRACALHKSPVATSDNVRTFDSFVMNKKIFKFDAVYVDEGLMVHTGLLNFALKISGCKKAFVFGDAKQIPFINRVMNFDYPKELRTLIVDNVERRYVTHRCPRDVTSFLNTIYKAAVATTSPVVHSVKAIKVSGAGILRPELTKIKGKIITFTQSDKQSLIKSGYNDVNTVHEIQGETFEETAVVRATPTPIGLIVRDSPHVLVALTRHTKAMVYYTVVFDAVTSIIVDVEKVDQSILTMFATTVPTK

The 129 kDa protein encoded by the attenuated CGMMV strain ONBM-32differs from the 129 kDa protein encoded by the wild type CGMMV Ontariostrain (SEQ ID NO:63) at least in that:

position 86 of SEQ ID NO:61 is serine (S, Ser);

position 480 of SEQ ID NO:61 is glycine (G, Gly);

position 534 of SEQ ID NO:61 is phenylalanine (F, Phe);

position 1092 of SEQ ID NO:61 is valine (V, Val); and

position 1124 of SEQ ID NO:61 is valine (V, Val).

The 186 kDa protein encoded by the attenuated CGMMV strain ONBM-32 hasthe following sequence.

(SEQ ID NO: 62) MANINEQINNQRDAAASGRNNLVSQLASKRVYDEAVRSLDHQDRRPKMNFSRVVSTEHTRLVTDAYPEFSISFTATKNSVHSLAGSLRLLELEYMMMQVPYGSPCYDIGGNYTQHLFKGRSYVHCCNPCLDLKDVARNVMYNDMVTQHVQRHKGSGGCRPLPTFQIDAFRRYDNSPCAVTCSDVFQECSYDFGSGRDNHAVSLHSIYDIPYSSIGPALHRKNVRVCYAAFHFSEALLLGSPVGNLNSIGAQFRVDGDDVHFLFSEESTLHYTHSLENIKLIVMRTYFPADDRYVYIKEFMVKRVDTFFFRLVRADTHMLHKSVGHYSKSKSEYFALNTPPIFQDKATFSVWFPEAKRKVLIPKFELSRFLSGNVKISRMLVDADFVHTIINHISTYDNKALVWKNVQSFVESIRSRVIVNGVSVKSEWNVPVDQLTDISFSIFLLVKVRKVQIELMSDKVVIEARGLLRRFADSLKSAVGGLGDCVYDALVQTGWFDTSSDELKVLLPEPFMTFSDYLEGMYEADAKIERESVFELLASGDDLFKKIDEIRNNYSGVEFDVEKFQEFCKELNVNPMLIGHVIEAIFSQKAGVTVTGLGTLSPEMGASVALSNTSVDTCEDMDVTEDMEDIVLMADKSHSYMSPEMARWADVKYDNNKGGLVEYKVGTSMTLPATWAEKGKAVLPLSGICVRKPQFSKPLDEEDDLRLSNMNFFKVSDLKLKKTITPVVYTGTIRERQMKNYIDYLSASLGSTLGNLERIVRSDWNGTEESMQTFGLYDCEKCKWLLLPAEKKHAWAVVLASDDTTRIIFLSYDESGSPIIDKRNWKRFAVCSETKVYSVIRSLEVLNKEAIVDPGVHITLVDGVPGCGKTAEIIARVNWKTDLVLTPGREAAAMIRRRACALHKSPVATSDNVRTFDSFVMNKKIFKFDAVYVDEGLMVHTGLLNFALKISGCKKAFVFGDAKQIPFINRVMNFDYPKELRTLIVDNVERRYVTHRCPRDVTSFLNTIYKAAVATTSPVVHSVKAIKVSGAGILRPELTKIKGKIITFTQSDKQSLIKSGYNDVNTVHEIQGETFEETAVVRATPTPIGLIVRDSPHVLVALTRHTKAMVYYTVVFDAVTSIIVDVEKVDQSILTMFATTVPTKXQLMQNSLYVHRDIFLPVSKTGFYTDMQEFYDRCLPGNSFVLNDFDAVTMRLRDNEFNLQPCRLTLSNLDPVPALVKSEAQNFLIPVLRTACERPRIPGLLENLVAMIKRNMNTPDLAGTVDITNMSISIVDNFFSSFVRDEVLLDHLDCVRASSIQSFSDWFSCQPTSAVGQLANFNFIDLPAFDTYMHMIKRQPKSRLDTSIQSEYPALQTIVYHLKVVNAVFGPVFKYLTTKFLSMVDSSKFFFYTRKKSEDLQEFFSDLSSHSDYEILELDVSKYDKSQSDFHFSIEMAIWEKLGLDDILAWMWSMGHKRTILQDFQAGIKTLIYYQRKSGDVTTFIGNTFIIAACVASMLPLDKCFKASFCGDDSLIYLPKGLEYPDIQATANLVWNFEAKLFRKKYGYFCGKYIIHHANGCIVYPDPLKLISKLGNKSLVGYEHVEEFRISLLDVAHSLFNGAYFHLLDDAIHELFPNAGGCSFVINCLCKYLSDKHLFRSLYIDVSK.

The 186 kDa protein encoded by the attenuated CGMMV strain ONBM-32differs from the 186 kDa protein encoded by the wild type CGMMV Ontariostrain (SEQ ID NO:64) at least in that:

position 86 of SEQ ID NO:62 is serine (S, Ser);

position 480 of SEQ ID NO:62 is glycine (G, Gly);

position 534 of SEQ ID NO:62 is phenylalanine (F, Phe);

position 1092 of SEQ ID NO:62 is valine (V, Val);

position 1124 of SEQ ID NO:62 is valine (V, Val);

position 1157 of SEQ ID NO:62 is aspartic acid (D, Asp);

position 1362 of SEQ ID NO:62 is leucine (L, Leu);

position 1397 of SEQ ID NO:62 is serine (S, Ser); and

position 1637 of SEQ ID NO:62 is histidine (H, His).

The coat protein encoded by the attenuated CGMMV strain ONBM-32 has thesequence of SEQ ID NO:32 and differs from the coat protein encoded bythe wild type CGMMV Ontario strain (SEQ ID NO:65) at least in thatposition 156 of SEQ ID NO:32 is valine (V, Val).

Example 3: Inoculation of Cucumber Plants with the Attenuated CGMMVONBM, ONBM-2 and ONBM-3 Strains

The attenuated CGMMV Ontario strains ONBM, ONBM-2 and ONBM-3 weretransformed into Agrobacterium tumefaciens strain EHA105 byelectroporation and used to inoculate the cotyledon of 1-2 week oldcucumber plants as described in Example 1. Two weeks after inoculation,plants were inspected for visible symptoms of CGMMV infection and leaftissue was collected for detection of virus infection. The attenuatedCGMMV Ontario strains ONBM, ONBM-2 and ONBM-3 were detected in leaftissue by real-time TaqMan reverse-transcription PCR (Chen et al.,Journal of Virological Methods (2008), 149: 326-329). As seen from theresults shown in FIGS. 3A to 3E, no symptoms were induced by inoculationwith ONBM (FIG. 3C), ONBM-2 (FIG. 3D) or ONBM-3 (FIG. 3E) while thetypical green mottle and mosaic symptoms were induced by inoculationwith wild-type CGMMV under laboratory greenhouse conditions (FIG. 3A).FIG. 3B shows leaves of a healthy cucumber plant grown as a control.

The attenuated CGMMV Ontario strains ONBM, ONBM-2 and ONBM-3 were testedfor the protection of cucumber plants from infection by wild-type CGMMV.Seven day old cucumber seedlings were inoculated with CGMMV Ontariostrains ONBM, ONBM-2 or ONBM-3 or were not inoculated, under laboratorygreenhouse conditions as described in Example 1. Two weeks afterinoculation, the plants were challenged with wild-type CGMMV Ontariostrain. As seen from the results shown in FIG. 4A to 4E, no symptomswere observed 4 weeks after challenge with the wild-type CGMMV on plantsinoculated with ONBM-2 (FIG. 4B) or ONBM-3 (FIG. 4C), and very mild orundetectable symptoms were observed 4 weeks after challenge with thewild-type CGMMV on plants inoculated with ONBM (FIG. 4A). In contrast,the typical green mottle and mosaic symptoms were observed inuninoculated plants challenged with wild-type CGMMV (FIG. 4D). FIG. 4Eshows leaves of an uninoculated and unchallenged healthy cucumber plant(control).

The attenuated CGMMV Ontario strains ONBM-2 and ONBM-3 were tested forthe protection of cucumber plants from natural infection by wild-typeCGMMV under commercial greenhouse conditions. Cotyledons of 1-2 week oldcucumber plants were inoculated with the strains ONBM-2 and ONBM-3 asdescribed in Example 1. Uninoculated cucumber plants were used as acontrol. As seen from the results presented in FIGS. 5A to 5E, novisible symptoms were observed on the cucumber plants after inoculationwith ONBM-2 or ONBM-3 for a period of 100 days (until the end ofproduction) in a commercial greenhouse. In contrast, uninoculated plantsshowed symptoms of CGMMV infection of leaves, showing green mottle andmosaic symptoms (FIG. 5A), and CGMMV infection of fruits (FIG. 5B andFIG. 5C). The cucumber fruits produced from the cucumber plantsinoculated with the attenuated strains ONBM-2 (FIG. 5D) or ONBM-3 (FIG.5E) were healthy, while the uninoculated cucumber plants produceddiseased and unmarketable cucumber fruits showing mosaic and soft (FIG.5B) or curling (FIG. 5C) symptoms. In addition, over a harvest period oftwo months, the cucumber fruit yield was increased by 12.7% during thesecond month of harvest for plants treated with the attenuated CGMMVONBM-3 strain compared with untreated cucumber plants exposed to naturalCGMMV infection in commercial greenhouse conditions.

Example 4: Inoculation of Cucumber Plants with the Attenuated CGMMVONAL-1, ONAL-2 and ONBM-32 Strains

The attenuated CGMMV Ontario strains ONAL-1, ONAL-2, and ONBM-32 weretransformed into Agrobacterium tumefaciens strain EHA105 byelectroporation. Agrobacterium tumefaciens containing mutant CGMMVstrains ONAL-1, ONAL-2, ONBM-32 and ONB (Example 2) were used toinoculate the cotyledon of 1-2 week old cucumber plants using the methoddescribed in Example 1. Two weeks after inoculation, plants wereinspected for visible symptoms of CGMMV infection and leaf tissue wascollected for detection of virus infection. The attenuated CGMMV Ontariostrains ONAL-1, ONAL-2, ONBM-32 and ONB were detected in leaf tissue byreal-time TaqMan reverse-transcription PCR (Chen et al., Journal ofVirological Methods (2008), 149: 326-329).

As seen from the results shown in FIGS. 6A to 6F, plants inoculated witheither mutant strains ONAL-1 (FIG. 6A) or ONAL-2 (FIG. 6B) underlaboratory greenhouse conditions showed mild symptoms compared withplants inoculated with the wild-type CGMMV Ontario strain, which showedtypical green mottle and mosaic symptoms (FIG. 6F). No symptoms wereobserved after inoculation with the mutant strain ONBM-32 (FIG. 6C). Thesymptoms observed after inoculation with mutant strain ONAL-2 (FIG. 6B)were milder than those observed after inoculation with mutant strainONAL-1 (FIG. 6A), and milder than those observed after inoculation withmutant strain ONB (FIG. 6D). FIG. 1E shows leaves of a healthy cucumberplant grown as a control.

The embodiments described herein are intended to be illustrative of thepresent compositions and methods and are not intended to limit the scopeof the present invention. Various modifications and changes consistentwith the description as a whole and which are readily apparent to theperson of skill in the art are intended to be included. The appendedclaims should not be limited by the specific embodiments set forth inthe examples, but should be given the broadest interpretation consistentwith the description as a whole.

What is claimed is:
 1. An attenuated strain of cucumber green mottlemosaic virus (CGMMV) comprising a genome, wherein the genome is apolyribonucleotide having a sequence functionally equivalent to avariant of SEQ ID NO:18, the sequence comprising at least one of thefollowing options a), b) and c): a) A at a position corresponding toposition 4969 of SEQ ID NO:18; b) U at a position corresponding toposition 3334 of SEQ ID NO: 18; and c) at least six nucleic acid basesselected from: A at a position corresponding to position 315 of SEQ IDNO: 18; G at a position corresponding to position 1498 of SEQ ID NO:18;U at a position corresponding to position 1660 of SEQ ID NO: 18; U at aposition corresponding to position 3430 of SEQ ID NO: 18; G at aposition corresponding to position 3528 of SEQ ID NO:18; U at a positioncorresponding to position 4144 of SEQ ID NO:18; U at a positioncorresponding to position 4248 of SEQ ID NO:18; and U at a positioncorresponding to position 6228 of SEQ ID NO:18.
 2. The attenuated strainaccording to claim 1 wherein the sequence of the polyribonucleotidecomprises one or more of A at the position corresponding to position4969 of SEQ ID NO: 18 and U at the position corresponding to position3334 of SEQ ID NO:
 18. 3. The attenuated strain according to claim 1wherein the sequence of the polyribonucleotide comprises: A at theposition corresponding to position 315 of SEQ ID NO: 18; G at theposition corresponding to position 1498 of SEQ ID NO: 18; U at theposition corresponding to position 1660 of SEQ ID NO:18; U at theposition corresponding to position 3430 of SEQ ID NO: 18; G at theposition corresponding to position 3528 of SEQ ID NO: 18; U at theposition corresponding to position 4144 of SEQ ID NO:18; U at theposition corresponding to position 4248 of SEQ ID NO: 18; and U at theposition corresponding to position 6228 of SEQ ID NO:
 18. 4. Theattenuated strain according to claim 3, wherein the sequence of thepolyribonucleotide further comprises one or more of A at the positioncorresponding to position 4969 of SEQ ID NO:18 and U at the positioncorresponding to position 3334 of SEQ ID NO:18.
 5. A composition forpreventing symptoms associated with infection by wild-type CGMMV in aplant or for increasing resistance of a plant to infection by wild-typeCGMMV, wherein the composition comprises an attenuated strain of CGMMVaccording to claim 1 and an agriculturally acceptable carrier.
 6. Anattenuated strain of cucumber green mottle mosaic virus (CGMMV)comprising a genome, wherein the genome is a polyribonucleotide encodinga 186 kDa protein having an amino acid sequence comprising at least oneof the following options a), b) and c): a) histidine at the positioncorresponding to position 1637 of SEQ ID NO:64; b) valine at theposition corresponding to position 1092 of SEQ ID NO:64; and c) at leastfive amino acid residues selected from: serine at the positioncorresponding to position 86 of SEQ ID NO:64; glycine at the positioncorresponding to position 480 of SEQ ID NO:64; phenylalanine at theposition corresponding to position 534 of SEQ ID NO:64; valine at theposition corresponding to position 1124 of SEQ ID NO:64; aspartic acidat the position corresponding to position 1157 of SEQ ID NO:64; leucineat the position corresponding to position 1362 of SEQ ID NO:64; andserine at the position corresponding to position 1397 of SEQ ID NO:64.7. The attenuated strain according to claim 6, wherein thepolyribonucleotide further encodes a coat protein having an amino acidsequence comprising valine at the position corresponding to position 156of SEQ ID NO:65.
 8. The attenuated strain according to claim 6, whereinthe amino acid sequence of the 186 kDa protein comprises at least oneamino acid residue selected from histidine at the position correspondingto position 1637 of SEQ ID NO:64 and valine at the positioncorresponding to position 1092 of SEQ ID NO:64.
 9. The attenuated strainaccording to claim 6, wherein the amino acid sequence of the 186 kDaprotein comprises at least five amino acid residues selected from:serine at the position corresponding to position 86 of SEQ ID NO:64;glycine at the position corresponding to position 480 of SEQ ID NO:64;phenylalanine at the position corresponding to position 534 of SEQ IDNO:64; valine at the position corresponding to position 1124 of SEQ IDNO:64; aspartic acid at the position corresponding to position 1157 ofSEQ ID NO:64; leucine at the position corresponding to position 1362 ofSEQ ID NO:64; and serine at the position corresponding to position 1397of SEQ ID NO:64.
 10. The attenuated strain according to claim 9, whereinthe amino acid sequence of the 186 kDa protein further comprises atleast one amino acid residue selected from histidine at the positioncorresponding to position 1637 of SEQ ID NO:64 and valine at theposition corresponding to position 1092 of SEQ ID NO:64.